neonatal-murine-cochlear-explant-technique-as-an-vitro-screening-tool

Neonatal Murine Cochlear Explant Technique as an <em>In Vitro</em> Screening Tool in Hearing Research

While there have been remarkable advances in hearing research over the past few decades, there is still no cure for Sensorineural Hearing Loss (SNHL), a ...

a-unified-methodological-framework-for-vestibular-schwannoma-research

A Unified Methodological Framework for Vestibular Schwannoma Research

Vestibular schwannomas are the most common neoplasms of the cerebellopontine angle, making up 6-8% percent of all intracranial growths. Though these ...

Massachusetts Eye and Ear

Harvard Program in Speech and Hearing Bioscience and Technology

Harvard Medical School

We hypothesize that the severity of hearing loss (HL) associated with sporadic vestibular schwannomas (VS) is correlated with tumor secretion of proteins with ototoxic or otoprotective potential.

Sporadic vestibular schwannomas associated with good hearing secrete higher levels of fibroblast growth factor 2 than those associated with poor hearing irrespective of tumor size.

Etiologies for many inner ear disorders, including autoimmune inner ear disease, sudden sensorineural hearing loss, and Meniere's disease, remain unknown. Indirect evidence suggests an immune-mediated process involving an allergic or autoimmune mechanism. We examined whether known immunogenic proteins share sequence similarity with inner ear proteins, which may lead to cross-reactivity and detrimental immune activation. Comprehensive bioinformatic analyses of primary sequences of intact and mutated proteins associated with human hearing loss and all proteins known to be expressed in the human inner ear were compared with all immune epitopes in the Immune Epitope Database. The exact match and basic local alignment search tool computational algorithms identified 3036 and 106 unique epitope matches, respectively, the majority of which were infectious epitopes. If validated in future clinical trials, these candidate immune epitopes in the inner ear would be potential novel targets for diagnosis and treatment of some inner ear disorders and the resulting hearing loss.

Mining immune epitopes in the inner ear.

Given the presence of a pathological immune response in sporadic vestibular schwannoma (sVS), this study aims to explore the roles of aspirin in minimizing sVS growth in vivo.

Aspirin intake correlates with halted growth of sporadic vestibular schwannoma in vivo.

Primary culture of human Schwann cells (SCs) and vestibular schwannoma (VS) cells are invaluable tools to investigate SC physiology and VS pathobiology, and to devise effective pharmacotherapies against VS, which are sorely needed. However, existing culture protocols, in aiming to create robust, pure cultures, employ methods that can lead to loss of biological characteristics of the original cells, potentially resulting in misleading biological findings. We have developed a minimally manipulative method to culture primary human SC and VS cells, without the use of selective mitogens, toxins, or time-consuming and potentially transformative laboratory techniques. Schwann cell purity was quantified longitudinally using S100 staining in SC cultures derived from the great auricular nerve and VS cultures followed for 7 and 12 weeks, respectively. SC cultures retained approximately ≥85% purity for 2 weeks. VS cultures retained approximately ≥80% purity for the majority of the span of 12 weeks, with maximal purity of 87% at 2 weeks. The VS cultures showed high level of biological similarity (68% on average) to their respective parent tumors, as assessed using a protein array featuring 41 growth factors and receptors. Apoptosis rate in vitro negatively correlated with tumor volume. Our results, obtained using a faster, simplified culturing method than previously utilized, indicate that highly pure, primary human SC and VS cultures can be established with minimal manipulation, reaching maximal purity at 2 weeks of culture. The VS cultures recapitulate the parent tumors' biology to a great degree, making them relevant models to investigate VS pathobiology.

Primary culture of human Schwann and schwannoma cells: improved and simplified protocol.

Vestibular schwannomas (VSs) are the most common tumors of the cerebellopontine angle. Significant clinical need exists for pharmacotherapies against VSs. Motivated by previous findings that immunohistochemical expression of cyclooxygenase 2 (COX-2) correlates with VS growth rate, we investigated the role of COX-2 in VSs and tested COX-2 inhibiting salicylates against VSs. COX-2 was found to be aberrantly expressed in human VS and primary human VS cells in comparison with control human nerve specimens and primary Schwann cells (SCs), respectively. Furthermore, levels of prostaglandin E2, the downstream enzymatic product of COX-2, were correlated with primary VS culture proliferation rate. Because COX-2 inhibiting salicylates such as aspirin are well tolerated and frequently clinically used, we assessed their repurposing for VS. Changes in proliferation, cell death, and cell viability were analyzed in primary VS cultures treated with aspirin, sodium salicylate, or 5-aminosalicylic acid. These drugs neither increased VS cell death nor affected healthy SCs. The cytostatic effect of aspirin in vitro was in concurrence with our previous clinical finding that patients with VS taking aspirin demonstrate reduced tumor growth. Overall, this work suggests that COX-2 is a key modulator in VS cell proliferation and survival and highlights salicylates as promising pharmacotherapies against VS.

Nonsteroidal anti-inflammatory medications are cytostatic against human vestibular schwannomas.

Vestibular schwannoma (VS), the fourth most common intracranial tumor, arises from the Schwann cells of the vestibular nerve. Although several pathways have been independently implicated in VS pathobiology, interactions among these pathways have not been explored in depth. We have investigated the potential cross-talk between hepatocyte growth factor (HGF) and vascular endothelial growth factor-A (VEGF-A) in human VS, an interaction that has been described in other physiological and pathological cell types. We affirmed previous findings that VEGF-A signaling is aberrantly upregulated in VS, and established that expression of HGF and its receptor cMET is also significantly higher in sporadic VS than in healthy nerves. In primary human VS and Schwann cell cultures, we found that VEGF-A and HGF signaling pathways modulate each other. siRNAs targeting cMET decreased both cMET and VEGF-A protein levels, and siRNAs targeting VEGF-A reduced cMET expression. Additionally, siRNA-mediated knockdown of VEGF-A or cMET and pharmacologic inhibition of cMET decreased cellular proliferation in primary human VS cultures. Our data suggest cross-talk between these 2 prominent pathways in VS and highlight the HGF/cMET pathway as an additional important therapeutic target in VS.

Interplay between VEGF-A and cMET signaling in human vestibular schwannomas and schwann cells.

Vestibular schwannomas (VSs), the most common tumors of the cerebellopontine angle, arise from Schwann cells lining the vestibular nerve. Pharmacotherapies against VS are almost non-existent. Although the therapeutic inhibition of inflammatory modulators has been established for other neoplasms, it has not been explored in VS. A bioinformatic network analysis of all genes reported to be differentially expressed in human VS revealed a pro-inflammatory transcription factor nuclear factor-kappa B (NF-κB) as a central molecule in VS pathobiology. Assessed at the transcriptional and translational level, canonical NF-κB complex was aberrantly activated in human VS and derived VS cultures in comparison to control nerves and Schwann cells, respectively. Cultured primary VS cells and VS-derived human cell line HEI-193 were treated with specific NF-κB siRNAs, experimental NF-κB inhibitor BAY11-7082 (BAY11) and clinically relevant NF-κB inhibitor curcumin. Healthy human control Schwann cells from the great auricular nerve were also treated with BAY11 and curcumin to assess toxicity. All three treatments significantly reduced proliferation in primary VS cultures and HEI-193 cells, with siRNA, 5 μM BAY11 and 50 μM curcumin reducing average proliferation (±standard error of mean) to 62.33% ± 10.59%, 14.3 ± 9.7%, and 23.0 ± 20.9% of control primary VS cells, respectively. These treatments also induced substantial cell death. Curcumin, unlike BAY11, also affected primary Schwann cells. This work highlights NF-κB as a key modulator in VS cell proliferation and survival and demonstrates therapeutic efficacy of directly targeting NF-κB in VS.

Preclinical validation of anti-nuclear factor-kappa B therapy to inhibit human vestibular schwannoma growth.

Vestibular schwannomas (VSs) are the most common tumours of the cerebellopontine angle. Ninety-five percent of people with VS present with sensorineural hearing loss (SNHL); the mechanism of this SNHL is currently unknown. To establish the first model to study the role of VS-secreted factors in causing SNHL, murine cochlear explant cultures were treated with human tumour secretions from thirteen different unilateral, sporadic VSs of subjects demonstrating varied degrees of ipsilateral SNHL. The extent of cochlear explant damage due to secretion application roughly correlated with the subjects' degree of SNHL. Secretions from tumours associated with most substantial SNHL resulted in most significant hair cell loss and neuronal fibre disorganization. Secretions from VSs associated with good hearing or from healthy human nerves led to either no effect or solely fibre disorganization. Our results are the first to demonstrate that secreted factors from VSs can lead to cochlear damage. Further, we identified tumour necrosis factor alpha (TNFα) as an ototoxic molecule and fibroblast growth factor 2 (FGF2) as an otoprotective molecule in VS secretions. Antibody-mediated TNFα neutralization in VS secretions partially prevented hair cell loss due to the secretions. Taken together, we have identified a new mechanism responsible for SNHL due to VSs.

Secreted Factors from Human Vestibular Schwannomas Can Cause Cochlear Damage.

Vestibular schwannoma (VS) is a tumor of the vestibular nerve that transmits balance information from the inner ear to the brain. Sensorineural hearing loss occurs in 95% of patients with these tumors, but the cause of this loss is not well understood. We posit a role of VS-secreted extracellular vesicles (EVs) as a major contributing factor in cochlear nerve damage.

ArticleTotal : 2	Year
Publication title Cited by 25	2017
Publication title Cited by 16	2017

Article	Year
Sporadic vestibular schwannomas associated with good hearing secrete higher levels of fibroblast growth factor 2 than those associated with poor hearing irrespective of tumor size. Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology\| PubMed ID: 23512073	2013
Mining immune epitopes in the inner ear. Otolaryngology--head and neck surgery : official journal of American Academy of Otolaryngology-Head and Neck Surgery\| PubMed ID: 24367049	2014
Aspirin intake correlates with halted growth of sporadic vestibular schwannoma in vivo. Otology & neurotology : official publication of the American Otological Society, American Neurotology Society [and] European Academy of Otology and Neurotology\| PubMed ID: 24448296	2014
Primary culture of human Schwann and schwannoma cells: improved and simplified protocol. Hearing research\| PubMed ID: 24910344	2014
Nonsteroidal anti-inflammatory medications are cytostatic against human vestibular schwannomas. Translational research : the journal of laboratory and clinical medicine\| PubMed ID: 25616959	2015
Interplay between VEGF-A and cMET signaling in human vestibular schwannomas and schwann cells. Cancer biology & therapy\| PubMed ID: 25692621	2015
Preclinical validation of anti-nuclear factor-kappa B therapy to inhibit human vestibular schwannoma growth. Molecular oncology\| PubMed ID: 25891780	2015
Secreted Factors from Human Vestibular Schwannomas Can Cause Cochlear Damage. Scientific reports\| PubMed ID: 26690506	2015
Extracellular vesicles derived from human vestibular schwannomas associated with poor hearing damage cochlear cells. Neuro-oncology\| PubMed ID: 27194145	2016

Sonam Dilwali

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