Principal Investigator University of Louisville, United States
Introduction: With an average survival of 15 months, glioblastoma (GBM) is one of the deadliest cancers and progresses rapidly despite aggressive chemotherapy and radiation. GBM progression is driven by a proneural to mesenchymal transition (PMT), whereby cells become increasingly invasive and therapy-resistant. The activation of this transition is not well understood, but emerging data suggest that biophysical cues can activate these changes independent of other stimuli. As it is well established that the tumor microenvironment contains a multitude of biophysical cues that can regulate cell fate and cancer progression, several of these inputs are manifest in late-stage cancer and may not be the primary driver of PMT. Mechanical cues that develop in the early stages of cancer progression likely underlie the emergence of mesenchymal GBM cells; however, these mechanisms are much less understood. Recent reports have identified intratumoral compression as a significant driver of cancer malignancy. Intratumoral compression is an early-stage mechanobiological cue that develops as a new primary tumor expands against the surrounding tissue, experiencing a normal force back that will increase as the tumor grows. The impact of compression in GBM is unknown, and its effect on regulating PMT remains unclear. Thus, the objective of this study is to assess the role of compressive stress on GBM mesenchymal transition and malignancy. The identification of the effect of compressive stress on GBM primary tumors may enable new therapeutic avenues for this devastating disease.
Materials and
Methods: Bulk GBM cells (U251) were cultured (DMEM with 10% FBS, 1% Penicillin Streptomycin, 1% Sodium Pyruvate, 1% Non-essential amino acids). Cells were seeded at 10,000 per cm2 on tissue culture-treated 0.4 µm pore 24 mm transwells for 12 hours. A 2% agarose disc is placed over each cell monolayer inside the transwell. The compressed conditions then have the addition of a cup and the respective number of tungsten carbide discs for the specific pressure application. The cell monolayers were compressed for 72 hours, after which they were fixed (4% Formalin with 1% Triton), lysed (RIPPA with 1% Protease/Phosphatase inhibitor), or lifted via Trypsin with 10% EDTA to be re-seeded for motility or invasion assays. For protein analysis, the lysates were normalized via BCA and then analyzed via western blot. For the immunostaining, the cells were incubated with primary antibody Gamma H2X anti-rabbit, then secondary Goat-anti rabbit and DAPI, before being imaged via Nikon Confocal microscopy at 60x. For motility, the cells were plated on tissue culture-treated plastic and imaged every 15 minutes over 24 hours. The cells were also seeded on a 3 µm pore tissue culture-treated transwell for 16 hours before being fixed via 4% Formalin with 1% Triton. The nucleus was stained via Propidium Iodide before imaging on a Nikon Epi-fluorescent scope.
Results, Conclusions, and Discussions: Our data reveal that compression promotes PMT, migration, and invasion. We first show that cells compressed within our system display elongated nuclei with a reduction in circularity and an increase in aspect ratio (Figure 1.A,B). After 72 h of compression at 400 Pa and 600 Pa, we found that ZEB1 expression was increased in both conditions, supporting mesenchymal transitions (Figure 1.C). We then assessed the functional changes associated with compressive stress. After cells were compressed for 72 hours, they were analyzed for migration potential with a random 2D motility assay. We found that compressed cells at 400 Pa and 600 Pa exhibited significant increases in cell speed and persistence (Figure 1.D,E). We then conducted a transwell invasion assay and found that 600 Pascals of compressive stress showed the highest number of cell invasion compared to control and 400 Pa groups (Figure 1.F). This data suggests that there may be a “dose-dependent” effect of compressive stress on cell invasion potential. Lastly, preliminary staining indicates compression also leads to elevated DNA damage, which is known to promote cancer cell malignancy (Figure 1.G). Collectively, our data provides novel evidence that elevated compressive stress leads to mesenchymal transitions and increases in migration and invasion potential in GBM.
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