(D-135) Development of a matrix metalloproteinase-9 activity based sensor to classify vestibular schwannomas

Introduction:: Vestibular schwannoma (VS) is the fourth most common intracranial neoplasm and most common tumor in the cerebellopontine angle, with a prevalence of 1 in 2000 adults¹. Patients with VS develop substantial morbidities including hearing loss which affects 95% of patients, tinnitus, dizziness, brainstem compression, hydrocephalus, and even death. VS occurs either sporadically or as a part of neurofibromatosis type 2 (NF2), a profoundly debilitating tumor disposition syndrome whose hallmark is bilateral VS. There is no cure for VS and no FDA-approved drugs. Microsurgery remains as the only option for tumor removal but is associated with tremendous risks such as deafness, facial paralysis, brain injury and incomplete tumor resection requiring additional procedures, especially when the tumor is aggressive and adherent to the brainstem and cranial nerves. Previous studies have implicated immune cell infiltration and protease activation in the pathogenesis of certain aggressive subtypes of VS^2,3. However, whether these molecular changes can be leveraged to classify adherent from non-adherent VS has yet to be explored. Here, we comprehensively characterized the tumor microenvironment (TME) of VS and identified matrix metalloproteinase 9 (MMP-9), a protease aberrantly upregulated in adherent VS. We also developed and optimized a FRET-based sensor that rapidly detected MMP-9 proteolytic activity, both in patient plasma and in fresh tumor specimens obtained from microsurgery. Taken together, findings from this study could lead to the development of tumor classifying biomarkers and improve surgical treatment outcomes for patients with VS.

Materials and Methods:: Patients with VS undergoing microsurgical resection were included in the study and tumors and plasma were collected intraoperatively. Relevant preoperative and postoperative clinical data was queried. Wild-type murine Schwann cells (WT-MSC) from mouse sciatic nerves and syngeneic mouse Schwann cell line deficient in merlin (genetic deletion of the NF2 gene using Cre-Lox)⁴ (MD-MSC) were implanted in nude mice to establish tumor allografts and the expression of 100 genes involved in ECM remodeling, protease activation and immune cell infiltration were analyzed using multiplexed transcriptomic gene expression profiling. Top gene candidates were validated in murine and human VS cell lines using qRT-PCR.
Immunohistochemical staining of candidate genes were performed in fresh frozen and paraffin-embedded, formalin fixed human VS specimens. The expression of each biomarker was correlated with clinical findings, such as the degree of peritumoral adhesions present intraoperatively. To design a sensor of in vivo protease activity in VS, a library of peptides bearing protease-cleavage motifs and paired FRET fluorophore/quencher was screened against recombinant proteases. The top performing sensor with the highest sensitivity and specificity was then applied to VS cell culture conditioned media and tumor secretions from tumors harvested during surgery. The ability of the sensor to spatially localize protease activity in VS was also evaluated in frozen tumor sections using in situ zymography.

Results, Conclusions, and Discussions:: Tumors from 28 patients were included, 16 (57.1%) were highly adherent and 12 tumors were non-adherent. Multiplexed gene expression of murine VS allografts identified 38 genes significantly upregulated (p=0.05) and 25 genes that were downregulated (p=0.05). Notably, several proteases were enriched in MD-MSC tumor allografts compared to WT-MSCs. Hierarchical clustering identified MMP-9 as the most significantly upregulated protease in MD-MSC by over 5-fold (Fig. 1A, p=0.05), which is also upregulated in murine (over 15-fold) and human vestibular schwannoma cell lines. In adherent human VS, MMP-9 was highly enriched in the tumor parenchyma compared to non-adherent VS (Fig. 1B). To develop a sensor of MMP9 activity, we designed 26 fluorogenic peptides bearing FRET fluorophore/quencher pairs and screened them against 12 recombinant metallo- and cysteine- proteases (Fig. 1C). Time-elapsed fluorescence dequenching studies were performed and the specificity of protease cleavage was probed with select small molecule inhibitors of MMP-2/9 activity. We identified S8 peptide sensor that showed the highest specificity by over 85% and sensitivity (V₀ 134 RFU/min) for detecting recombinant MMP-9 activity. S8 readily detected MMP-9 activity in conditioned media from human VS cell line (V₀ 22 RFU/min), primary human VS tumor secretions (V₀ 47 RFU/min), and plasma from VS patients (V₀ 125 RFU/min, Fig 1D), demonstrating the potential of MMP-9 as a secreted VS biomarker detectable in plasma. Finally, S8 was applied as an in-situ zymography probe to detect MMP-9 activity within the parenchyma of VS. Compared to a control peptide sensor that was not cleaved by MMPs, S8 sensor was able to accurately localize to areas of elevated protease activity (over 5-fold increase in FRET fluorescence vs. control peptide), and co-localized with vestibular schwannoma cells (Fig 1E).

The TME of schwannomas is characterized by dysregulated protease activity, notably elevated MMP-9 expression, which is also highly enriched in aggressive VS that are adherent to the brainstem and cranial nerves. A library of fluorogenic FRET peptides was screened to identify a peptide that senses MMP-9 activity in both tumor secretions and in plasma of VS patients, and localizes to areas of elevated MMP-9 activity in human VS ex vivo.



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