Single-cell mechanical analysis reveals viscoelastic similarities between astrocytes and glioblastoma cells

Introduction: The physical attributes of the host organ can significantly affect cancer spread. For tumors originating outside of the brain, it has been reported that cancer cells become softer and more mobile than normal cells in response to local mechanical forces, promoting their migration and metastasis away from the primary tumor. However, these mechanobiological phenomena have yet to be explored in the brain. Here, we aimed to compare the viscoelastic properties of human U87 glioblastoma (GBM) cells and their normal counterpart, immortalized human astrocytes (IHAs) using a parallel-plate flow chamber. Furthermore, given the essential role of actin in cellular mechanical behavior and adhesion in cell-cell and cell-substrate interactions, we sought to quantify actin density and alignment as well as adhesion energy using fluorescence microscopy and atomic force microscopy, respectively. Indeed, an understanding of GBM cell mechanics may lead to the identification of novel disease mechanisms, biomarkers, and therapeutic strategies to improve treatment outcomes.

Materials and

Methods: We employed our previously established fluid shear stress assay technique to investigate and compare the viscoelastic properties (namely, stiffness, viscosity, and relaxation time) of normal IHAs and GBM cells. We specifically used a parallel-plate flow shear system to subject single cells to physiological levels of fluid shear stress (FSS) present in the brain microenvironment (4 Pa) and monitored the resulting deformation in real-time using a camera-coupled optical microscope. Following FSS exposure, we utilized the Davis 10.1.2 software to generate strain-time profiles of individual cells via digital image correlation analysis. We then characterized the nuclear and cytoplasmic viscoelastic properties of all cells by fitting their respective deformation profiles with a three-parameter generalized Maxwell model. We also quantified actin cytoskeleton density and alignment in both cell types by imaging phalloidin-stained IHAs and GBM cells with a Keyence BZ-X810 widefield microscope. To measure actin density, we quantified the mean actin stain intensity of individual cells using the Leica fluorescence image processing software LAS X. Furthermore, we performed fiber alignment analysis using the MATLAB-based software CT-FIRE. Fiber angles were normalized to the circular mean angle for each cell and combined for statistical analysis. Additionally, we used atomic force microscopy to determine the adhesion energies of individual cells from each cell type.

Results, Conclusions, and Discussions: In total, we analyzed the viscoelastic properties of 84 U87 cells and 96 IHAs. Comparative analysis revealed that the nuclear and cytoplasmic viscoelastic properties of U87 cells and IHAs are not statistically significantly different (p > 0.05) (Fig. 1). Therefore, our study suggests that no distinct mechanical differences exist between GBM cells and their normal, healthy counterpart. However, we did observe statistically significant differences (p < 0.05) between the nuclear and cytoplasmic stiffness and relaxation times of IHAs, but not U87 GBM cells (Fig. 2). The difference between nuclear and cytoplasmic viscosity in both cell types was statistically insignificant.
The actin density and alignment of 29 U87 cells and 32 IHAs was analyzed via fluorescent microscopy. Fluorescent intensity analysis revealed that IHAs and GBM cells have similar levels of actin density as there were no statistically significant differences between the fluorescent intensity per cell area of both cell types (Fig. 3). On the other hand, analysis of the actin fibers showed that the actin fibers of IHAs are typically more organized and aligned relative to U87 cells. Interestingly, preliminary atomic force microscopy results indicate that IHAs and GBM cells have a statistically significant difference in adhesion energy with IHAs having a moderately higher value (Fig. 4).
Overall, our results suggest that GBM cells may exhibit unique behaviors not seen in other extracranial cancer cell types. Notably, given the important role cellular mechanics plays in cancer cell migration, as well as the low mechanical demands and high fluid content in the brain, malignant cells may adopt unique brain-specific mechanical properties via inherent (genetic) and externally-driven (microenvironmental) mechanisms. This may explain why metastasis in GBM patients is rare and conventional treatment options are unsuccessful, although future studies directly evaluating this connection are required. The impact of experimental parameters such as substrate stiffness is also a subject of future studies as tuning them may reveal mechanical differences not captured in this study.

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