(H-309) The Role of Intra-tumoral Compressive Stress on Glioblastoma Malignancy

Glioblastoma (GBM) is one of the deadliest cancers with a survival rate of 15 months after diagnosis. This is due in part to the rapid invasion of GBM cells to the surrounding tissue despite aggressive treatment. These invading cells are thought to have undergone a proneural-mesenchymal transition (PMT), which heightens invasion potential and therapy resistance. The mechanism by which this transition happens is poorly understood, but studies have indicated that mechanical cues in the GBM tumor microenvironment play a significant role in this process. Although many studies have examined mechanical cues outside of the tumor such as substrate stiffness, little is known about the impact of mechanical cues inside the tumor. As tumor expand, the intra-tumoral stress rises and applies compressive stresses to the tumor cells within the tumor. New studies have indicated that increased compression contributes to the development of a leader-follower relationship and is potentially a pro-malignant signal. The effect of increased compression and its influence on the malignant nature of this cancer is largely understudied. Here, we explore the effect of compressive stress on PMT and cancer malignancy through examination of cell proliferation, migration, and phenotype.

Bulk GBM tumor cells (U251, U87) were cultured in DMEM with 10% FBS, 1% Penicillin Streptomycin, 1% Sodium Pyruvate, and 1% Non-essential Amino Acids. Cells were seeded at on the transwells at 500k cells per cm² for 12 hours before being compressed for 48 hours (Greiner Bio-One). After compression, the cells were either fixed via 4% formalin with 1% triton and stained for Ki-67, Zeb1, and DAPI. Nuclear shape, nuclear size, fluorescent colocalization, and fluorescent intensity were assessed. For motility assays, cells were lifted from the transwell then seeded for 12 hours before time lapse microscopy in which migration speed and persistence was assessed. A t-test was used to determine statistical significance between groups.

Compressive stress of 1000 Pa via the compression system (Figure 1A) over 48 hours promotes nuclear deformation (Figure 1B), cells become less round (Figure 1C) and larger in size (Figure 1D). After 48 hours of compression, cells migrate significantly faster (Figure 1E) and are more persistent than the non-treated condition (Figure 1F). Furthermore, compressed cells also showed down regulation of the proliferation marker Ki-67 (Figure 1G) and upregulation of ZEB1 (Figure 1H), a transcription factor that promotes mesenchymal cell nature. These results suggests that compression is linked to a shift towards malignant nature and supports PMT.

            We demonstrate here that compression promotes the behaviors seen in mesenchymal cells, like speed and persistence, and also increases mesenchymal transcription factor ZEB1. This experimental strategy provides insight into the role of compression in promoting the aggressive behavior of GBM tumor cells and can be used further to elucidate the mechanism underlying this change. Additionally, this approach can be applied to investigate mechanisms important in developmental biology, stem cell biology, and fibrotic disease. Further investigation may provide new approaches for prognostic tests and therapeutics.