Vestibular schwannomas (VSs) are the most common tumours of the cerebellopontine angle. Ninety-five percent of people with VS present with sensorineural hearing loss (SNHL); the mechanism of this SNHL is currently unknown. To establish the first model to study the role of VS-secreted factors in causing SNHL, murine cochlear explant cultures were treated with human tumour secretions from thirteen different unilateral, sporadic VSs of subjects demonstrating varied degrees of ipsilateral SNHL. The extent of cochlear explant damage due to secretion application roughly correlated with the subjects’ degree of SNHL. Secretions from tumours associated with most substantial SNHL resulted in most significant hair cell loss and neuronal fibre disorganization. Secretions from VSs associated with good hearing or from healthy human nerves led to either no effect or solely fibre disorganization. Our results are the first to demonstrate that secreted factors from VSs can lead to cochlear damage. Further, we identified tumour necrosis factor alpha (TNFα) as an ototoxic molecule and fibroblast growth factor 2 (FGF2) as an otoprotective molecule in VS secretions. Antibody-mediated TNFα neutralization in VS secretions partially prevented hair cell loss due to the secretions. Taken together, we have identified a new mechanism responsible for SNHL due to VSs.

Hypothesis We hypothesize that the severity of hearing loss (HL) associated with sporadic vestibular schwannomas (VS) is correlated with tumor secretion of proteins with ototoxic or otoprotective potential. Background Since the recognition that HL associated with VS is not solely due to compression of the auditory nerve, elucidating the mechanism by which VS cause HL has been an important task. We previously showed that VS stratified by hearing have differential gene expression. We now focus on identifying differentially expressed proteins in tumor secretions. Methods Fresh surgical specimens of VS were incubated in sterile PBS at 37°C to collect secretions. The specimens were divided into a group associated with good hearing (GH, word recognition ≥70% and pure-tone average ≤30 dB, n=11) or poor hearing (PH, n=10). The groups were compared using a customized cytokine array. Statistically significant results were verified with ELISA on a different set of secretions (n=8 for GH and n=10 for PH group). Results Of the 37 molecules we studied, 9 were significantly expressed in secretions from VS compared to secretions from control nerves. Secretion of fibroblast growth factor 2 (FGF2) was 3.5-fold higher in VS associated with GH versus PH based on cytokine array analysis (p=0.02), which was validated with ELISA. Conclusions This study highlights FGF2, a mitogen known to protect the auditory nerve, as a potential tumor-secreted mediator of hearing protection in VS. If FGF2's significant role in hearing protection in patients with VS is validated, then FGF2 could be utilized as a biomarker for HL in VS and therapeutic targeting of the FGF2 signaling pathway may reduce HL due to VS.

Vestibular schwannomas (VSs) are the most common tumors of the cerebellopontine angle. Significant clinical need exists for pharmacotherapies against VSs. Motivated by previous findings that immunohistochemical expression of cyclooxygenase 2 (COX-2) correlates with VS growth rate, we investigated the role of COX-2 in VSs and tested COX-2 inhibiting salicylates against VSs. COX-2 was found to be aberrantly expressed in human VS and primary human VS cells in comparison to control human nerve specimens and primary Schwann cells (SCs), respectively. Further, levels of prostaglandin E2, the downstream enzymatic product of COX-2, correlated with primary VS culture proliferation rate. Because COX-2 inhibiting salicylates such as aspirin are well-tolerated and frequently clinically used, we assessed their repurposing for VS. Changes in proliferation, cell death and cell viability were analyzed in primary VS cultures treated with aspirin, sodium salicylate (NaSal) or 5-aminosalicylic acid (5-ASA). These drugs did not increase VS cell death nor affect healthy SCs. The cytostatic effect of aspirin in vitro was in concurrence with our previous clinical finding that VS patients taking aspirin demonstrate reduced tumor growth. Overall, this work suggests that COX-2 is a key modulator in VS cell proliferation and survival, and highlights salicylates as promising pharmacotherapies against VS.

Our results suggest a potential therapeutic role of aspirin in inhibiting sVS growth.

This is the first report on EVs derived from VSs and on the capacity of EVs from VSs from patients with hearing loss to selectively damage cochlear cells, thereby identifying a potential novel mechanism of VS-associated sensorineural hearing loss.

Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology.

Vestibular schwannomas (VSs), the most common tumors of the cerebellopontine angle, arise from Schwann cells lining the vestibular nerve. Pharmacotherapies against VS are almost non-existent. Although the therapeutic inhibition of inflammatory modulators has been established for other neoplasms, it has not been explored in VS. A bioinformatic network analysis of all genes reported to be differentially expressed in human VS revealed a pro-inflammatory transcription factor nuclear factor-kappa B (NF-κB) as a central molecule in VS pathobiology. Assessed at the transcriptional and translational level, canonical NF-κB complex was aberrantly activated in human VS and derived VS cultures in comparison to control nerves and Schwann cells, respectively. Cultured primary VS cells and VS-derived human cell line HEI-193 were treated with specific NF-κB siRNAs, experimental NF-κB inhibitor BAY11-7082 (BAY11) and clinically relevant NF-κB inhibitor curcumin. Healthy human control Schwann cells from the great auricular nerve were also treated with BAY11 and curcumin to assess toxicity. All three treatments significantly reduced proliferation in primary VS cultures and HEI-193 cells, with siRNA, 5 μM BAY11 and 50 μM curcumin reducing average proliferation (± standard error of mean) to 62.33% ± 10.59 %, 14.3 ± 9.7 %, and 23.0 ± 20.9 % of control primary VS cells, respectively. These treatments also induced substantial cell death. Curcumin, unlike BAY11, also affected primary Schwann cells. This work highlights NF-κB as a key modulator in VS cell proliferation and survival and demonstrates therapeutic efficacy of directly targeting NF-κB in VS.

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