Non-invasive monitoring of vestibular schwannoma progression by in-situ MMP-9 localization using peptide functionalized gold nanoparticles

Introduction: Vestibular schwannoma (VS) is one of the most common brain tumors and causes significant disabilities which impact hearing, balance, a person’s ability to communicate, and can even result in brainstem compression and hydrocephalus. Unfortunately, there is currently no cure for VS and no FDA-approved drugs, leaving surgical resection as the only curative option when tumor growth is detected by MRI [1]. The unavailability of effective tools to understand VS progression on the molecular level hinders early VS detection and disease management. While tumor growth is one indicator of disease severity, tumor size alone does not accurately reflect its underlying biology. Furthermore, MRI has limitations include limited accessibility and high cost. Proteases, particularly matrix metalloproteinase-9 (MMP-9), play a crucial role by remodeling the tumor extracellular matrix and promote angiogenesis during VS progression [2]. Due to protease dysregulation, they have been identified as the potential target for tumor diagnosis and therapy. Currently, there is no way to detect in-situ protease activity in VS. This lack of a robust detection method hinders the ability to fully understand tumor biology and classification. Developing better in-situ protease activity assays holds significant promise for improving early VS diagnosis.

Materials and

Methods: A custom peptide library was screened against 11 recombinant matrix metalloproteinases, with the peptide most specific to MMP-9 cleavage (S8) selected for further development. These peptides were flanked by a fluorophore and quencher, allowing cleavage by MMP-9 to generate a fluorescent signal based on FRET. Gold nanoparticles (AuNPs) were synthesized and functionalized with different lengths (0.1-20 kDa) of polyethylene glycol (PEG). These PEGylated AuNPs were conjugated with S8 peptides at different valencies (1:5-1:15). The peptide-AuNPs were applied to syngeneic mouse Schwann cell line deficient in merlin (genetic deletion of the NF2 gene using Cre-Lox) (MD-MSC) cells, biofluids from patients with VS undergoing surgical resection, and followed by in-situ zymography on frozen VS tissue sections. MD-MSCs were implanted in nude mice to establish tumor allografts [3]. Tumors were incubated with AuNPs conjugated with either MMP-9 cleavable and a non-cleavable peptide containing d-isomers. The specific peptide consists of a charged sequence masked by a peptide substrate sequence that prevents it from binding the tissue until cleaved by localized target proteases. The characterizations were done using Fluorescence resonance energy transfer (FRET), UV-vis spectroscopy, dynamic light scattering (DLS), in-situ zymography and Immunofluorescence. Quantification and visualization were performed with ImageJ.

Results, Conclusions, and Discussions: Results and discussion: Our study developed a method to detect MMP-9, crucial for tumor progression and classification. High MMP-9 levels were found in VS tumors (Fig. 1 B). ROC analysis identified both MMP9 and tumor volume as a VS classifying biomarker (AUCMMP9+vol=0.91, Sensitivity 75%, Specificity 96%) (Fig. 1 C). Peptide library identified a specific peptide (S8) efficiently cleaved by MMP-9 and with minimal cleavage by other proteases as shown by unsupervised hierarchical clustering (Fig. 1 D). S8 conjugation to AuNPs was characterized by UV-spectra and DLS (Fig E-G). Optimal PEG length (5K) and peptide density (1:12) were determined based on maximum cleavage efficiency by MMP-9 while minimizing background (Fig. 1 H,I). Biofluid samples from VS patients undergoing surgical tumor resection (plasma and tumor secretions) showed high levels of protease activity, potentially allowing tumor classification (Fig. J). In MD-MSC cells, S8 strongly localized MMP-9 activity compared to a control peptide matched in overall peptide charge (S23) (Fig. 2A). A charged peptide (S8Z9) conjugated with PEGylated-AuNPs was used for in situ zymography which uses electrostatic interaction for tissue binding upon cleavage whereas a non-cleavable, d-stereoisomer, dS8Z9 was used as control (Fig.2 B,C p=0.004). The nanoparticles presence in tissues allows for visualization of MMP-9 spatial localization within tumor tissues. We observed higher fluorescence intensity in areas with high cell density or adjacent to blood vessels (Fig. 2 D), indicating angiogenesis. Healthy nerve tissue showed minimal to no fluorescence signal compared to VS (Fig. 2 E), demonstrating nanoprobe’s ability as a disease classifier to distinguish between healthy and vestibular schwannomas. The nanoprobe also effectively demonstrated a strong correlation between fluorescence intensity and tumor growth (Fig. 2F). In tumors with even minimum size, we observed significant changes in fluorescence, suggesting its potential for early and increased tumor detection.


Conclusion: This study presents a novel method for detection of MMP-9 activity in-situ in VS, using a specific protease-sensitive nanoprobe design which effectively differentiates tumors based on protease levels and can even detect small size tumors. It holds promise for early VS detection, classification, MMP-9 localization within tumors with the potential of photothermal therapy for tumor treatment.

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

References:
1. Steve E.J. Connor MRCP, FRCR. Imaging of the Vestibular Schwannoma. ClinicalKey. 2021; 31(4):451-471.
2. Møller, M.N., Werther, K., Nalla, A., Stangerup, S.-E., Thomsen, J., Bøg-Hansen, T.C., Nielsen, H.J. and Cayé-Thomasen, P. (2010), Angiogenesis in vestibular schwannomas: Expression of extracellular matrix factors MMP-2, MMP-9, and TIMP-1†. The Laryngoscope, 120: 657-662.
3. Giovannini M, Robanus-Maandag E, van der Valk M, et al. Conditional biallelic Nf2 mutation in the mouse promotes manifestations of human neurofibromatosis type 2. Genes & development. 2000; 14(13):1617-1630