Matrix metalloproteinase 9: an emerging biomarker for classification of adherent vestibular schwannoma

Introduction: Vestibular schwannoma (VS) is a prevalent intracranial tumor commonly found in the cerebellopontine angle, affecting 1 in 2000 adults1. This tumor presents significant treatment challenges, particularly when it is adherent to the brainstem or cranial nerves, often requiring complex surgical interventions with a high risk of complications such as hearing loss, facial paralysis, brain injury, and incomplete tumor resection. Currently, there are no FDA-approved drugs for VS, with microsurgery being the only option for tumor removal. Previous studies have implicated immune cell infiltration and protease activation in the pathogenesis of certain aggressive subtypes of VS2. Recent studies have identified MMP9 as a protease involved in extracellular matrix remodeling (ECM) associated with tumor progression and invasion. Elevated levels of matrix metalloproteinase-9 (MMP9) has been linked to aggressive tumor in VS, suggesting its potential as a biomarker for identifying tumors with poor prognostic outcomes and as a potential therapeutic target to improve surgical treatment outcomes3.

Materials and

Methods: Patients with VS undergoing microsurgical resection were included and tumors and plasma were collected intraoperatively. Preoperative and postoperative clinical data were analyzed. Wild-type murine Schwann cells (WT-MSC) from mouse sciatic nerves and a syngeneic mouse Schwann cell line deficient in merlin (MD-MSC) due to genetic deletion of the NF2 gene (genetic deletion of the NF2 gene using Cre-Lox)4 were used to establish tumor allografts in nude mice. The expression of 100 genes related to ECM, protease activation, and immune cell infiltration was examined using multiplex transcriptomic profiling. Transcriptomic profiling of murine schwannoma allografts was performed. Differentially expression genes (DEGs) in MD-MSC versus WT-MSC was validated using qRT-PCR. The spatial expression of MMP9 during schwannoma progression was validated using immunofluorescence staining. Additionally, human VS samples were collected prospectively for immunohistochemical analysis to assess MMP-9 spatial expression patterns. ELISA were employed to measure MMP-9 levels in murine cell and human VS primary cultures. The functional role of MMP-9 in cellular invasion was evaluated using transwell invasion assays. To determine blot the in vitro potential therapeutic strategy to mitigate schwannoma aggressiveness, the efficacy of inhibiting the eukaryotic initiation factor 4F (eIF4F) complex using Silvestrol, which binds to the 5’-UTR of MMP-9, was evaluated in MD-MSCs. Finally, to assess the therapeutic potential of MMP9 inhibitors in vivo, mice bearing schwannoma allografts were treated with MMP9-IN-I (3 mg/kg).

Results, Conclusions, and Discussions: We profiled genes with known roles in ECM remodeling with a focus on proteases (Fig 1A). Two-dimensional unsupervised hierarchical clustering of MD-MSC and WT-MSCs demonstrated two clusters (Fig 1B). There was an enrichment of metalloproteases with MMP9 being the most abundantly expressed protease (Fig 1C). To examine the spatial expression of MMP9 during mouse schwannoma progression, we stained tumors 1 week after implantation (small) and 3 weeks after implantation when tumors reached 1 cm in diameter (large). MMP9 initially concentrated around SOX-10+ schwannoma cells. As the tumor grew, MMP9 expression was more widespread. MMP9 expression within the tumor parenchyma increased by 5-fold in large tumors (Fig 2A, B, p< 0.0001). Adherent human VS primary cultures secreted higher levels of MMP9 than non-adherent VS (Fig 2C,49.7 vs.13.8 ng/mL, p=0.014). To compare MMP9 expression around the tumor vasculature in adherent and non-adherent tumors, we identified regions containing CD31+ vessels and queried the co-localization of MMP9 expression on consecutive histological sections (Figure 2D). In non-adherent VS, 30% CD31+ vessels colocalized with MMP9 (Fig 2E, p< 0.0002). By contrast, 70% of adherent VS endothelium also showed perivascular MMP9 expression (Figure 3E, p< 0.0002). To investigate the functional effect of MMP9 on cell invasion, we observed that inhibition of the eIF4F complex effectively reduced MMP-9 activity in schwannoma cells, indicating a potential therapeutic role (Fig 3A, B p< 0.05). In vitro assays demonstrated that MMP-9 significantly enhances schwannoma cell invasion, particularly under TNF-α stimulation (Fig 3C, p< 0.0002). Finally, MMP9-IN-I treatment significant reduced tumor burden by 50% with minimal systemic toxicity (Fig 3D, E, p< 0.0001).

Conclusions: We identify MMP9 as a highly active protease during vestibular schwannoma growth and in adherent VS, representing a potential classifying biomarker. Having the knowledge of tumor MMP9 levels preoperatively could improve patient outcome by predicting intraoperative tumor adherence. In the future, developing technologies to detect MMP9 and therapeutics against MMP9 in adherent VS could represent a breakthrough in the management of these tumors which otherwise have no cure.

Acknowledgements (Optional): National Institute on Deafness and Other Communication Disorders (NIDCD) grant 1K08DC020761-01 (YR) and the American Neurotology Society Research Grant (YR).

References
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