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A cerebellopontine angle mouse model for the investigation of tumor biology, hearing, and neurological function in NF2-related vestibular schwannoma

Abstract

Neurofibromatosis type II (NF2) is a disease that lacks effective therapies. NF2 is characterized by bilateral vestibular schwannomas (VSs) that cause progressive and debilitating hearing loss, leading to social isolation and increased rates of depression. A major limitation in NF2 basic and translational research is the lack of animal models that allow the full spectrum of research into the biology and molecular mechanisms of NF2 tumor progression, as well as the effects on neurological function. In this protocol, we describe how to inject schwannoma cells into the mouse brain cerebellopontine angle (CPA) region. We also describe how to apply state-of-the-art intravital imaging and hearing assessment techniques to study tumor growth and hearing loss. In addition, ataxia, angiogenesis, and tumor–stroma interaction assays can be applied, and the model can be used to test the efficacy of novel therapeutic approaches. By studying the disease from every angle, this model offers the potential to unravel the basic biological underpinnings of NF2 and to develop novel therapeutics to control this devastating disease. Our protocol can be adapted to study other diseases within the CPA, including meningiomas, lipomas, vascular malformations, hemangiomas, epidermoid cysts, cerebellar astrocytomas, and metastatic lesions. The entire surgical procedure takes ~45 min per mouse and allows for subsequent longitudinal imaging, as well as neurological and hearing assessment, for up to 2 months.

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Fig. 1: Surgical procedure for stereotactic injection in CPA model.
Fig. 2: Confirmation of CPA tumor location and evaluation of tumor growth.
Fig. 3: Surgery and tumor implantation techniques do not affect hearing or neurological function.
Fig. 4: CPA tumors induce hearing loss and ataxia in mice.
Fig. 5: Intravital microscopy imaging of NF2−/− tumors in the CPA model.

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Data availability

The data obtained using this protocol are available at https://doi.org/10.1073/pnas.1719966115.

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Acknowledgements

We thank S.M. Chin and V. Askoxylakis for their superb technical support. This study was supported by a Department of Defense (DoD) New Investigator Award (W81XWH-16-1-0219, L.X.); an American Cancer Society Research Scholar Award (RSG-12-199-01-TBG, L.X.); the Children’s Tumour Foundation Drug Discovery Initiative (L.X.); an Ira Spiro Award (L.X.); a Simeon J. Fortin Charitable Foundation Postdoctoral Fellowship (M.R.N.); a DoD BCRP Postdoctoral Fellowship (W81XWH-14-1-0034, M.R.N.); grants NCI P01-CA080124, P50-CA165962, R01-CA129371, R01-CA208205, and U01-CA 224348; an Outstanding Investigator Award (R35-CA197743, R.K.J.); the Lustgarten Foundation; the Ludwig Center at Harvard; the National Foundation for Cancer Research; the Gates Foundation (R.K.J.); NIDCD grant R01DC015824 (K.M.S.); the Bertarelli Foundation (K.M.S.); the Nancy Sayles Day Foundation (K.M.S.); the Lauer Tinnitus Research Center (K.M.S.); the Barnes Foundation (K.M.S.); and the Zwanziger Foundation (K.M.S.).

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Authors

Contributions

J.C., L.D.L., R.K.J., S.R.P., K.M.S., and L.X. designed the research; J.C., L.D.L., Y.S., J.R., N.M., L.W., M.R.N., J.W.C., N.Z., Y.Z., X.G., T.F., S.R., and P.H. performed the research; J.C., L.D.L., J.R., N.M., M.R.N., J.W.C., K.M.S., and L.X. analyzed the data; and L.D.L., Y.S., J.R., N.M., R.K.J., S.R.P., K.M.S., and L.X. wrote the paper.

Corresponding authors

Correspondence to Konstantina M. Stankovic or Lei Xu.

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Competing interests

R.K.J. has received consultant fees from Merck, Ophthotech, Pfizer, Sun Pharma Advanced Research Corporation (SPARC), SynDevRx, and XTuit; owns equity in Enlight, Ophthotech, SynDevRx, and XTuit; serves on the board of directors of XTuit and the boards of trustees of Tekla Healthcare Investors, Tekla Life Sciences Investors, Tekla Healthcare Opportunities Fund, and Tekla World Healthcare Fund; and received honorarium for speaking at AMGEN. No funding or reagents from these companies were used in these studies. The remaining authors declare no competing interests.

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Key references using this protocol

Zhao, Y. et al. Proc. Natl. Acad. Sci. USA 115, E2077–E2084 (2018): https://doi.org/10.1073/pnas.1719966115

Jensen, J. B., Lysaght, A. C., Liberman, M. C., Qvortrup, K. & Stankovic, K. M. PLoS ONE 10, e0125160 (2015): https://doi.org/10.1371/journal.pone.0125160

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Chen, J., Landegger, L.D., Sun, Y. et al. A cerebellopontine angle mouse model for the investigation of tumor biology, hearing, and neurological function in NF2-related vestibular schwannoma. Nat Protoc 14, 541–555 (2019). https://doi.org/10.1038/s41596-018-0105-7

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