Mechanical Compression Promotes Pro-Aggressive Phenotypic Shifts in Vestibular Schwannoma

Introduction: : Neurofibromatosis type 2 (NF2) is an aggressive tumor predisposition syndrome with no cure. A hallmark feature of NF2 is the development of bilateral vestibular schwannomas (VS) that originate from the cochleovestibular cranial nerve, which leads to deafness, vertigo, brainstem compression and even death. VS originates within the rigid bony internal auditory canal and experiences significant mechanical compression. How these forces influence tumor behavior is unknown, as current cell and animal models do not recapitulate this unique mechanical microenvironment. This knowledge gap prevents the identification of mechanoresponsive therapeutic targets. Here, we investigate the effect of extrinsic mechanical compression on VS, focusing on characteristics including cell motility, cytoskeletal remodeling and activation of Yes-associated protein (YAP) signaling.

Materials and

Methods: : Mouse NF2-/- schwannoma cells were cultured on 0.4 µm transwell membranes and underwent uniaxial compression (0.1-0.16 kPa) using 3D printed cylindrical weights (Formlabs Form 2 SLA) for 6-24 hours to simulate solid stress levels in brain. Cells underwent immunofluorescence staining and were visualized by confocal microscopy to extract biophysical data including nuclear morphologies, markers of proliferation, apoptosis and cell-cell adhesion, cytoskeleton remodeling, and activation of YAP signaling. Expression profiles were compared between compressed and uncompressed cells. Nuclear morphology was analyzed using circularity index and the nuclear-to-cytoplasmic ratio of YAP expression was quantified (ImageJ). The expression of genes involved in mechanotransduction and YAP signaling were analyzed by qRT-PCR. Migration capacity was assessed using a scratch wound assay. Statistical significance was evaluated using unpaired t-tests with a threshold of p < 0.05.

Results, Conclusions, and Discussions:: Schwannoma cells exhibited varying degrees of proliferation and apoptosis under compression (Fig. 1A). Mechanical compression at 0.13 kPa for 17 hours resulted in a 5-fold increase in expression of cleaved caspase-3 and a 2.5-fold decrease in Ki67 (Fig. 1B, p< 0.0001). Morphologically, compressed cells displayed significantly reduced circularity and re-organization of actin stress fibers compared to uncompressed cells (0.5 ± 0.1 vs. 0.9 ± 0.1; p < 0.0001, Fig. 1C), adopting a more elongated cell shape which was associated with enhanced cell motility. Increased N-cadherin recycling and vesicular punctate expression pattern was observed with higher degrees of mechanical compression (p < 0.05, Fig. 1D), suggesting enhanced intercellular adhesion critical for collective cell migration under increased mechanical stress. Schwannoma cells demonstrated a progressive increase in nuclear-to-cytoplasmic YAP expression ratio when compressed from 0.1kPa to 0.16 kPa, with a maximum of 3.7-fold increase at 0.16 kPa (p < 0.0001, Fig. 1E), signifying YAP nuclear translocation and transcriptional activation. Compressed VS cells demonstrated elevated migratory capacity and a significant increase in wound closure after 0.13kPa compression for 17 hours (p < 0.01, Fig. 1F). Amongst the mechanosensitive genes examined (TAZ, TEAD, CTCF, PIEZO1, TRPV4), TRPV4 mRNA expression was the most upregulated in compressed cells by nearly 2-fold (p=0.0286, Fig. 1G). Our study provides the first evidence that mechanical compression alters the phenotype of vestibular schwannoma, promotes apoptosis, cell-cell adhesion and migration through YAP signaling. Relieving solid stress may represent a novel therapeutic target by reducing tumor migratory potential. Future studies should investigate the efficacy of YAP inhibitors in VS.

Acknowledgements and/or References (Optional):: This work was supported by the National Institute on Deafness and Other Communication Disorders (NIDCD) grant 1K08DC020761-01 (Y.R.) and the American Neurotology Society Research Grant (Y.R.)