(K-431) Stiffness-Based Cell Sorting Microfluidic Device to Examine Stability and Heritability of Cancer Cell Mechanotypes

Introduction:: Metastasis, or the spread of cancer cells from their primary tumor location to distant sites in the body, causes a majority of cancer-related deaths. The metastatic and migratory potential of tumor cells is related to their mechanical properties, such as cell stiffness. However, previous studies of cell stiffness have been limited to observing cell mechanics as an effect of rather than a potential driving force of metastasis. If we could establish a causal link between cell stiffness and metastatic potential, the direct modulation of cell mechanics could constitute a therapeutic strategy to slow or stop the metastatic spread of cancer cells.

To explore this potential causal link, I first separated stiff and soft cell subpopulations with a microfluidic stiffness-based cell sorting device (Figure 1a)^1,2. This technology consists of a microchannel with multiple diagonal ridges that hang down from the top plane of the device that compress cells as they flow through the 8 micron gap (Figure 1b). Cells that are either highly deformable, smaller, more viscous, or less adhesive are able to squeeze under the ridges and follow the hydrodynamic forces of the flowing buffer around the cells to travel towards a specific device outlet. Alternatively, cells that are stiffer, larger, less viscous, or more adhesive resist compression and are moved by the diagonal ridges perpendicular to the fluid flow toward different device outlets (Figure 1c). In this way, a heterogenous population of cells can be sorted into subpopulations with different mechanical properties.


Materials and Methods:: A heterogenous breast cancer cell line, MDA-MB-231, was sorted into mechanical subpopulations with the microfluidic device. To optimize the operation of the device, cells were tracked during high-speed videos (2000 frames per second) as they flowed through the device at different volumetric flow rates and analyzed with a combination of the ImageJ plugin TrackMate and custom python-based code to determine the amount of deflection of cells to the outlets of interest for each flow rate (Figure 2a). Atomic force microscopy and micropipette aspiration were used to test the mechanical properties of the parental and sorted populations. After allowing time for cells to divide and labeling each subpopulation with a different Cell Tracker dye, the cells were re-sorted and flow cytometry was used to identify whether the labeled cells initially sorted into the stiff or soft subpopulations re-sort into the same stiff or soft subsets following cell division. Serial sorting was utilized to make stable subpopulations of stiff and soft cells and to investigate the extent of heritability of cell stiffness.


Results, Conclusions, and Discussions:: To optimize fractionation of the cell line into mechanical sub-populations, we tested multiple volumetric flow rates for the microfluidic device. Reducing the flow rate results in an increased proportion of cells deflected to outlets 4 and 5 (Figure 2b). Sorting relies on the balance between the hydrodynamic forces causing cells to follow fluid streamlines through the device with the normal force a cell experiences as it squeezes underneath a ridge. As the flow rate decreases, the normal force outweighs the hydrodynamic forces and cells that are more resistant to deformation are pushed along the ridges towards outlets 4 and 5. At 40 µL/min per channel, we observed a high degree of sorting by size (Figure 2c). For the first time with this device design, staged sorting was used to first sort cells by size and then by stiffness. The device was first operated at 40 µL/min to sort cells by their size. Cells from one of the size-binned outlets were then re-sorted at a slower flow rate to sort by stiffness. In this way, the population of homogenously sized cells were further fractionated with an increased proportion of cells deflected to outlets 4 and 5 at slower flow rates in the second stage of sorting (Figure 3a and 3b). Atomic force microscopy was used to compare the stiffness of cells deflected to outlet 2 and 5, resulting in an observed enrichment of stiff cells in outlet 5 (Figure 3c). Stiffness sorting was further optimized to increase the purity and efficiency of removing stiff cells from the fluid stream using serial sorting, resulting in increased sensitivity and specificity. Finally, using Cell Tracker dyes enabled tracking of cell stiffness changes hours and days after sorting, establishing a mechanism to investigate the stability and heritability of cell stiffness tracking over time and after cell division and passaging (Figure 3d). Understanding cell mechanotype as a transient state or an inherent property gives us insight into the plasticity of cancer cells’ mechanical state, which can eventually lead to the development of targeted therapies to slow or stop metastasis through the modulation of cancer cell mechanics.

Acknowledgements (Optional): : This research project was supported by the Keck Foundation.


References (Optional): : 1. Wang, G., Mao, W., Byler, R., Patel, K., Henegar, C., Alexeev, A., and Sulchek, T. (2013). Stiffness Dependent Separation of Cells in a Microfluidic Device. PLOS ONE 8, e75901. 10.1371/journal.pone.0075901.

2. Young, K.M., Shankles, P.G., Chen, T., Ahkee, K., Bules, S., and Sulchek, T. (2022). Scaling microfluidic throughput with flow-balanced manifolds to simply control devices with multiple inlets and outlets. Biomicrofluidics 16, 034104. 10.1063/5.0080510.