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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Data availability
The data obtained using this protocol are available at https://doi.org/10.1073/pnas.1719966115.
References
Evans, D. G. et al. A clinical study of type 2 neurofibromatosis. Q. J. Med. 84, 603–618 (1992).
Plotkin, S. R., Merker, V. L., Muzikansky, A., Barker, F. G. 2nd & Slattery, W. 3rd Natural history of vestibular schwannoma growth and hearing decline in newly diagnosed neurofibromatosis type 2 patients. Otol. Neurotol. 35, e50–56 (2014).
West, J. S., Low, J. C. & Stankovic, K. M. Revealing hearing loss: a survey of how people verbally disclose their hearing loss. Ear Hear. 37, 194–205 (2016).
Kim, S. Y. et al. Severe hearing impairment and risk of depression: a national cohort study. PLoS ONE 12, e0179973 (2017).
Plotkin, S. R. et al. Hearing improvement after bevacizumab in patients with neurofibromatosis type 2. N. Engl. J. Med. 361, 358–367 (2009).
Zhao, Y. et al. Targeting the cMET pathway augments radiation response without adverse effect on hearing in NF2 schwannoma models. Proc. Natl. Acad. Sci. USA 115, E2077–E2084 (2018).
Tannous, B. A. Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo. Nat. Protoc. 4, 582–591 (2009).
Bovenberg, M. S., Degeling, M. H. & Tannous, B. A. Enhanced Gaussia luciferase blood assay for monitoring of in vivo biological processes. Anal. Chem. 84, 1189–1192 (2012).
Gao, X. et al. Anti-VEGF treatment improves neurological function and augments radiation response in NF2 schwannoma model. Proc. Natl. Acad. Sci. USA 112, 14676–14681 (2015).
Guyenet, S. J. et al. A simple composite phenotype scoring system for evaluating mouse models of cerebellar ataxia. J. Vis. Exp. 2010, e1787 (2010).
Jain, R. K., Munn, L. L. & Fukumura, D. Dissecting tumour pathophysiology using intravital microscopy. Nat. Rev. Cancer 2, 266–276 (2002).
Vakoc, B. J., Fukumura, D., Jain, R. K. & Bouma, B. E. Cancer imaging by optical coherence tomography: preclinical progress and clinical potential. Nat. Rev. Cancer 12, 363–368 (2012).
Brown, E. B. et al. In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nat. Med. 7, 864–868 (2001).
Dilwali, S., Landegger, L. D., Soares, V. Y., Deschler, D. G. & Stankovic, K. M. Secreted factors from human vestibular schwannomas can cause cochlear damage. Sci. Rep. 5, 18599 (2015).
Soares, V. Y. et al. Extracellular vesicles derived from human vestibular schwannomas associated with poor hearing damage cochlear cells. Neuro Oncol. 18, 1498–1507 (2016).
Wong, H. K. et al. Anti-vascular endothelial growth factor therapies as a novel therapeutic approach to treating neurofibromatosis-related tumors. Cancer Res. 70, 3483–3493 (2010).
Bonne, N. X. et al. An allograft mouse model for the study of hearing loss secondary to vestibular schwannoma growth. J. Neurooncol. 129, 47–56 (2016).
Dinh, C. T. et al. A xenograft model of vestibular schwannoma and hearing loss. Otol. Neurotol. 39, e362–e369 (2018).
McClatchey, A. I., Saotome, I., Ramesh, V., Gusella, J. F. & Jacks, T. The Nf2 tumor suppressor gene product is essential for extraembryonic development immediately prior to gastrulation. Genes Dev. 11, 1253–1265 (1997).
Giovannini, M. et al. Schwann cell hyperplasia and tumors in transgenic mice expressing a naturally occurring mutant NF2 protein. Genes Dev. 13, 978–986 (1999).
Giovannini, M. et al. Conditional biallelic Nf2 mutation in the mouse promotes manifestations of human neurofibromatosis type 2. Genes Dev. 14, 1617–1630 (2000).
Kalamarides, M. et al. Nf2 gene inactivation in arachnoidal cells is rate-limiting for meningioma development in the mouse. Genes Dev. 16, 1060–1065 (2002).
Gehlhausen, J. R. et al. A murine model of neurofibromatosis type 2 that accurately phenocopies human schwannoma formation. Hum. Mol. Genet. 24, 1–8 (2015).
Messing, A. et al. P0 promoter directs expression of reporter and toxin genes to Schwann cells of transgenic mice. Neuron 8, 507–520 (1992).
Gutmann, D. H. & Giovannini, M. Mouse models of neurofibromatosis 1 and 2. Neoplasia 4, 279–290 (2002).
McClatchey, A. I. et al. Mice heterozygous for a mutation at the Nf2 tumor suppressor locus develop a range of highly metastatic tumors. Genes Dev. 12, 1121–1133 (1998).
Neff, B. A. et al. Cochlear implantation in the neurofibromatosis type 2 patient: long-term follow-up. Laryngoscope 117, 1069–1072 (2007).
Meijer, E. F., Baish, J. W., Padera, T. P. & Fukumura, D. Measuring vascular permeability in vivo. Methods Mol. Biol. 1458, 71–85 (2016).
McCann, C. M. et al. Combined magnetic resonance and fluorescence imaging of the living mouse brain reveals glioma response to chemotherapy. Neuroimage 45, 360–369 (2009).
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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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.
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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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DOI: https://doi.org/10.1038/s41596-018-0105-7
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