This work was supported by Centro Grandi Strumenti Universit&#224; dell&#39;Insubria, Fondazione Comunitaria del Varesotto, Fondazione del Monte di Lombardia, and Fondazione Anna Villa e Felice Rusconi.

All the studies were performed according the Declaration of Helsinki (1975) with written informed consent and approved by the Ethical Committee of the University of Insubria in Varese.
1. Collection of the Samples
<ol>
	<li>Collect and divide all the human Formalin-Fixed Paraffin-Embedded (FFPE) samples into different subgroups according the WHO classification of Head and Neck Tumors14. 
	NOTE: Here we used the following samples: Normal sinonasal mucosa as control (10 samples); Inverted Papilloma (IP, ICD-O code 8121/1); Sinonasal ITAC (ICD-O code 8144/3, 32 samples); NITAC (ICD-O code 8140/3, 12 samples); Adenoid Cystic Carcinoma (ACC, ICD-O code 8200/3); Pleomorphic Adenoma (PA, ICD-O code 8941/3); ON (ICD-O code 9522/3, 13 samples); Poorly Differentiated Neuroendocrine Carcinoma (PDNEC, ICD-O code 8041/3, 19 samples); Neuroendocrine Tumor (NET, ICD-O code 8041/3).</li>
</ol>
2. Immunochemistry
<ol>
	<li>Deparaffinization and rehydration of sections
	<ol>
		<li>Heat the sample slides in an oven at 60 &#176;C for 30 min and rehydrate 3 &#181;m thick FFPE sections using an alcohol series to water.</li>
		<li>Briefly wash the slides in xylene for 10 min and repeat this step; wash the slides for 5 min in 100% alcohol and repeat this step using 95%, 85%, and 75% alcohol serially. Rinse the slides for 5 min in distilled water.</li>
	</ol>
	</li>
	<li>Blocking the endogenous activity
	<ol>
		<li>Block the endogenous activity by placing the slides in 3% aqueous hydrogen peroxide for 12 min.</li>
	</ol>
	</li>
	<li>Antigen retrieval
	<ol>
		<li>Perform antigen retrieval by treating with 10 mM Citrate Buffer (pH 6) for 10 min in a microwave treatment.</li>
	</ol>
	</li>
	<li>Incubation with primary antibody
	<ol>
		<li>Wash the sections in TBS buffer (pH 7.4), then add blocking solution for 10 min.</li>
		<li>Wash the sections in TBS buffer (pH 7.4), then add 0.2% Triton X.</li>
		<li>Incubate overnight at 4 &#176;C with goat anti-human OTX2 antibody diluted 1:100.</li>
	</ol>
	</li>
	<li>Incubation with secondary antibody
	<ol>
		<li>Incubate the sections for 1 h at room temperature with biotinylated rabbit anti-goat secondary antibody diluted 1:200 followed by ABC-peroxidase complex (see Tables of Materials).</li>
	</ol>
	</li>
	<li>Developing the immunoreaction
	<ol>
		<li>Develop the immunoreaction using 3,3&#39;-diaminobenzidine tetrahydrochloride and counterstain the nuclei with Harris Hematoxylin.</li>
	</ol>
	</li>
	<li>Mounting and imaging
	<ol>
		<li>Dehydrate the sections using a crescent alcohol-scale and clarify them using a clearing substance of terpene origin (see Table of Materials). Embed sections in mounting medium, place the sections on a microscope slide, and observe the sections through an optical microscope.</li>
	</ol>
	</li>
</ol>
3. RNA Extraction and Reverse-transcription
<ol>
	<li>RNA extraction
	<ol>
		<li>Extract RNA from the sections by performing the first step of immunoprecipitation protocol (see section 2.1) and keep the slides in distilled water. Overlap the unstained sections with the corresponding hematoxylin-eosin stained sections in order to identify fragments of interest.</li>
		<li>Perform the RNA extraction using a commercial RNA extraction kit for FFPE samples (see Table of Materials) and following manufacturer&#39;s instructions.</li>
	</ol>
	</li>
	<li>RNA reverse-transcription
	<ol>
		<li>Use a commercial kit to retro-transcribe RNA into cDNA (see Table of Materials) following the protocol. Retro-transcribe at least 1,000 ng of total RNA to perform several analyses.</li>
	</ol>
	</li>
</ol>
4. Real-time PCR and Data Analysis
<ol>
	<li>Real-time PCR
	<ol>
		<li>Perform quantitative real-time PCR analyses (qRT-PCR) with probe-based technology (see Table of Materials) and a thermal cycler.</li>
	</ol>
	</li>
	<li>Preparation of the PCR reaction mix
	<ol>
		<li>Prepare the PCR reaction mix using 12.5 &#181;L of probe-based master mix, 1.25 &#181;L each of OTX1, OTX2, and ACTB probes (see Table of Materials), 50 ng of cDNA, and nuclease-free water up to 25 &#181;L of total volume.</li>
		<li>Perform all the reactions in triplicate using the ACTB gene as the endogenous control to normalize gene expression levels.</li>
		<li>Centrifuge the plate at 1,109 x g for 3 min and store the plate protected from light at 4 &#176;C until the experiment.</li>
	</ol>
	</li>
	<li>Setting the thermal cycler
	<ol>
		<li>Set the thermal cycler profile with an initial hot start cycle at 50 &#176;C for 2 min and 95 &#176;C for 10 min, followed by 40 cycles at 95 &#176;C for 15 s and a final cycle at 60 &#176;C for 1 min.</li>
	</ol>
	</li>
	<li>Gene expression level analyses
	<ol>
		<li>Normalize the gene expression levels through the comparative cycle threshold (&#916;Ct) method using the ACTB gene as the endogenous control.</li>
		<li>Evaluate gene expression levels using the 2-&#916;Ct method and plot the results.</li>
	</ol>
	</li>
	<li>Statistical analyses
	<ol>
		<li>Perform statistical analyses using Student&#39;s t-Test, considering statistically significant results with p &#60; 0.05.</li>
	</ol>
	</li>
</ol>

<ol><li>Boncinelli, E., Simeone, A., Acampora, D., Gulisano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Homeobox+genes+in+the+developing+central+nervous+system%5BTitle%5D)+AND+%22Ann+genet%22%5BJournal%5D)">Homeobox genes in the developing central nervous system.</a> Ann genet. 36 (1), 30-37 (1993).</li>
<li>Omodei, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+the+Brain+Transcription+Factor+OTX1+Occurs+in+a+Subset+of+Normal+Germinal-Center+B+Cells+and+in+Aggressive+Non-Hodgkin+Lymphoma%5BTitle%5D)+AND+%22American+J.+Pathol%22%5BJournal%5D)">Expression of the Brain Transcription Factor OTX1 Occurs in a Subset of Normal Germinal-Center B Cells and in Aggressive Non-Hodgkin Lymphoma.</a> American J. Pathol. 175, 2609-2617 (2009).</li>
<li>Levantini, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unsuspected+role+of+the+brain+morphogenetic+gene+Otx1+in+hematopoiesis%5BTitle%5D)+AND+%22Proc.+Natl.+Acad.+Sci+USA%22%5BJournal%5D)">Unsuspected role of the brain morphogenetic gene Otx1 in hematopoiesis.</a> Proc. Natl. Acad. Sci USA. 100 (18), 10299-10303 (2003).</li>
<li>Larsen, K. B., Lutterodt, M. C., Mollgard, K., Moller, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Expression+of+the+homeobox+OTX2+and+OTX1+in+the+early+developing+human+brain%5BTitle%5D)+AND+%22J.+Histochem.+Cytochem%22%5BJournal%5D)">Expression of the homeobox OTX2 and OTX1 in the early developing human brain.</a> J. Histochem. Cytochem. 58, 669-678 (2010).</li>
<li>Abate-Shen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Deregulated+homeobox+gene+expression+in+cancer%3A+cause+or+consequence%3F%5BTitle%5D)+AND+%22Nat.+Rev.+Cancer%22%5BJournal%5D)">Deregulated homeobox gene expression in cancer: cause or consequence?</a> Nat. Rev. Cancer. 2, 777-785 (2002).</li>
<li>de Haas, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((OTX1+and+OTX2+expression+correlates+with+the+clinicopathologic+classification+of+medulloblastomas%5BTitle%5D)+AND+%22J.+Neuropathol.+Exp.+Neurol%22%5BJournal%5D)">OTX1 and OTX2 expression correlates with the clinicopathologic classification of medulloblastomas.</a> J. Neuropathol. Exp. Neurol. 65, 176-186 (2006).</li>
<li>Terrinoni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((OTX1+expression+in+breast+cancer+is+regulated+by+p53%5BTitle%5D)+AND+%22Oncogene%22%5BJournal%5D)">OTX1 expression in breast cancer is regulated by p53.</a> Oncogene. 30 (27), 3096-3103 (2001).</li>
<li>Yu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((OTX1+promotes+colorectal+cancer+progression+through+epithelial-mesenchymal+transition%5BTitle%5D)+AND+%22Biochem.+Biophys+Res+Commun%22%5BJournal%5D)">OTX1 promotes colorectal cancer progression through epithelial-mesenchymal transition.</a> Biochem. Biophys Res Commun. 44 (1), 1-5 (2014).</li>
<li>Glubrecht, D. D., Kim, J. H., Russel, L., Bamforth, J. S., Godbout, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Differential+CRX+and+OTX2+expression+in+human+retina+and+retinoblastoma%5BTitle%5D)+AND+%22J.+Neurochem%22%5BJournal%5D)">Differential CRX and OTX2 expression in human retina and retinoblastoma.</a> J. Neurochem. 111 (1), 250-263 (2009).</li>
<li>Coletta, R. D., Jedlicka, P., Gutierrez-Hartamn, A., Ford, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transcriptional+control+of+the+cell+cycle+in+mammary+gland+development+and+tumorigenesis%5BTitle%5D)+AND+%22J.+mammary+gland+Biol.+Neoplasia%22%5BJournal%5D)">Transcriptional control of the cell cycle in mammary gland development and tumorigenesis.</a> J. mammary gland Biol. Neoplasia. 9, 39-53 (2004).</li>
<li>Cordes, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Molecular+and+phenotypic+analysis+of+poorly+differentiated+sinonasal+neoplasms%3A+an+integrated+approach+for+early+diagnosis+and+classification%5BTitle%5D)+AND+%22Hum+Pathol%22%5BJournal%5D)">Molecular and phenotypic analysis of poorly differentiated sinonasal neoplasms: an integrated approach for early diagnosis and classification.</a> Hum Pathol. 40, 283-292 (2009).</li>
<li>Stelow, E. B., Bishop, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Update+from+the+4th+Edition+of+the+World+Health+Organization+Classification+of+Head+and+Neck+Tumours%3A+Tumors+of+the+Nasal+Cavity%2C+Paranasal+Sinuses+and+Skull+Base%5BTitle%5D)+AND+%22Head+Neck+Pathol%22%5BJournal%5D)">Update from the 4th Edition of the World Health Organization Classification of Head and Neck Tumours: Tumors of the Nasal Cavity, Paranasal Sinuses and Skull Base.</a> Head Neck Pathol. 11 (1), 3-15 (2017).</li>
<li>Llorente, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Sinonasal+carcinoma%3A+clinical%2C+pathological%2C+genetic+and+therapeutic+advances%5BTitle%5D)+AND+%22Nat.+Rev.+Clin.+Oncol%22%5BJournal%5D)">Sinonasal carcinoma: clinical, pathological, genetic and therapeutic advances.</a> Nat. Rev. Clin. Oncol. 11 (8), 460-472 (2014).</li>
<li>Sidransky, D. <a target="_blank" href="https://www.researchgate.net/profile/Lester_Thompson/publication/7200778_World_Health_Organization_classification_of_tumours_Pathology_and_genetics_of_head_and_neck_tumours/links/09e415102df86bd4a2000000.pdf">World health organization classification of tumors. Pathology and genetics of head and neck tumors</a>. 9, WHO Press. (2005).</li>
<li>Radulesku, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Endoscopic+surgery+for+sinonasal+tumors%3A+The+transcribriform+approach%5BTitle%5D)+AND+%22J+Stomatol+Oral+Maxillofac+Surg%22%5BJournal%5D)">Endoscopic surgery for sinonasal tumors: The transcribriform approach.</a> J Stomatol Oral Maxillofac Surg. 118 (4), 248-250 (2017).</li>
<li>Muller, P. A. J., Vousden, K. H. <a target="_blank" href="https://www.nature.com/articles/ncb2641">p53 mutations in cancer.</a> Nature cell biology. 15 (1), 2-8 (2013).</li>
<li>Holmila, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Profile+of+TP53+gene+mutations+in+sinonasal+cancer%5BTitle%5D)+AND+%22Mutat+Res%22%5BJournal%5D)">Profile of TP53 gene mutations in sinonasal cancer.</a> Mutat Res. 686 (1-2), 9-14 (2010).</li>
</ol>

The authors have nothing to disclose.

This study shows for the first time that, based on mRNA levels, the HB genes OTX1 and OTX2 are expressed in normal sinonasal mucosa and submucosal glands, inflammatory polyps, sinonasal Schneiderian papillomas, and in the different epithelial and neuroectodermal neoplasms, including squamous carcinomas, non-intestinal type sinonasal adenocarcinomas, salivary gland-type tumors, neuroendocrine neoplasms, and ONs.
Modifications and Troubleshooting:
To avoid RNA degradatio...

OTX HB genes are the vertebrate homologue of the Drosophila orthodenticle genes (otd) and they encode for transcription factors which are normally expressed during embryonic morphogenesis, but they can also be expressed in the adult organism with different functions. During embryonic development they control the specification of cell identity, cell differentiation, and the positioning of the body axis&#185;. The OTX family includes OTX1 and OTX2 genes which display different functions. OTX1 is involved in brain and sensory organ development. In the adult organism, it is expressed in sensory organs and is transcribed at low levels in the anterior lobe of the pituitary gland2; it also plays a role in hematopoiesis, being expressed in hematopoietic pluripotent and progenitor cells3. OTX2 is involved in the development of the rostral head and its translated protein acts as a morphogen because it generates a gradient through which other genes are activated or repressed in a spatio-temporal manner, thus contributing to cell proliferation and differentiation. In the adult organism, OTX2 is found exclusively in the choroid plexus and pineal gland4.
Mutations in OTX genes are often related to the appearance of human congenital, somatic, or metabolic defects. Gain or loss mutations in OTX genes could promote tumorigenesis if they are not able to properly control cellular growth and/or differentiation5. In leukemias and lymphomas as well as in many solid tumors (e.g., medulloblastomas6, aggressive non-Hodgkin lymphomas2, breast carcinomas7, colorectal cancers8, and retinoblastoma9), the deregulated expression of OTX HB genes is well documented10. In addition, OTX2 mutations have been demonstrated in cases of anophthalmia and microphtalmia11 due to the crucial role for this gene in the control of eye development.
In the context of solid neoplasms, the discovery of molecular and phenotypic markers is an important challenge for the diagnosis, classification, and treatment of several types of tumor11, including those that originate in the nasal cavity and paranasal sinuses. In fact, despite that these areas occupy only a modest anatomical space, mucosal epithelium, glands, soft tissues, bone, cartilage or neural/neuroectodermal, and hematolymphoid cells can be often the site for the origin of complex and histologically different groups of tumors. Different types of neoplasms involving the sinonasal tract present a variety of features that overcome what is usually seen in the upper aerodigestive tract or even throughout most parts of the body12.Sinonasal malignancies are rare and present an annual incidence of 1:100,000 inhabitants worldwide, and so this prevents studies regarding the pathways involved in the tumorigenesis and the testing of alternative treatment strategies.Despite this, the advances in imaging techniques, surgical approaches, and radiotherapy have improved the clinical management of sinonasal cancer.Moreover, the development of cell lines as well as animal models and cancer genetic profiling currently constitute the basis for the future targeted anticancer therapies13. To date, there are no reports regarding OTX1 and/or OTX2 expression in neoplasms of the nasal cavity, paranasal sinuses, and nasopharynx. Since we have previously observed that OTX1 and OTX2 are involved in breast cancer7, we wondered if these genes could be present not only in the normal nasal mucosa but also in tumors of the nasal cavity. To reach this goal we obtained from the Department of Pathology of the &#34;Ospedale di Circolo&#34; in Varese samples of normal mucosa, and nasal and sinonasal adenocarcinomas collected from 1985 to 2012 and classified according to the World Health Organization (WHO) classification of Head and Neck Tumors. We choose to analyze them through real-time PCR and immunohistochemistry analyses and we evaluated OTX1 and OTX2 expression to determine if they can be considered molecular markers for these types of tumors.

<table><tbody><tr><td>RecoverAll Total Nucleic Acid Isolation</td><td>Applied Biosystem</td><td>AM1975</td><td></td></tr><tr><td>High-Capacity cDNA Reverse Transcription Kit</td><td>Applied Biosystem</td><td>4368814</td><td></td></tr><tr><td>TaqMan Universal PCR Master Mix, no AmpErase UNG</td><td>Applied Biosystem</td><td>4326614</td><td></td></tr><tr><td>ABI Prism 7000&amp;nbsp;</td><td>Applied Biosystem</td><td>270001857</td><td></td></tr><tr><td>ACTB probe</td><td>Applied Biosystem</td><td></td><td>Out of production. Sequence protected by copyright</td></tr><tr><td>OTX1 probe</td><td>Applied Biosystem</td><td></td><td>Out of production. Sequence protected by copyright</td></tr><tr><td>OTX2 probe</td><td>Applied Biosystem</td><td></td><td>Out of production. Sequence protected by copyright</td></tr><tr><td>Acqueous Hydrogen Peroxide</td><td>Merk</td><td>1072090250</td><td></td></tr><tr><td>Citrate Buffer</td><td>Sigma-Aldrich</td><td>20276292</td><td></td></tr><tr><td>Triton</td><td>Sigma-Aldrich</td><td>101473728</td><td></td></tr><tr><td>Tris</td><td>Merk</td><td>108382</td><td></td></tr><tr><td>NaCl</td><td>Merk</td><td>106404</td><td></td></tr><tr><td>Goat Anti-OTX2 Antibody</td><td>Vector Laboratories</td><td></td><td>Out of production. Catalog number not available</td></tr><tr><td>Rabbit Anti-Goat Antibody</td><td>Vector Laboratories</td><td>BA5000</td><td></td></tr><tr><td>ABC-Peroxidase Complex</td><td>Vector Laboratories</td><td>PK6100</td><td></td></tr><tr><td>3,3&#039;-diaminobenzidine tetrahydrocloride (DAB)</td><td>Sigma-Aldrich</td><td>D5905</td><td></td></tr><tr><td>Harris Hematoxylin</td><td>Bioptica</td><td>0506004/L</td><td></td></tr><tr><td>Pertek</td><td>Kaltek SRL</td><td>1560</td><td></td></tr><tr><td>BioClear</td><td>Bioptica</td><td>W01030799</td><td></td></tr></tbody></table>

identification of otx1 and otx2 as two possible molecular markers for sinonasal carcinomas and olfactory neuroblastomas

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations occurring in these genes are often related to tumorigenesis in human. No data are available today regarding the possible correlation between OTX genes and tumors of the nasal cavity. The aim of this work is to understand if OTX1 and OTX2 can be considered as molecular markers in the development of nasal tumors. We selected nasal and sinonasal adenocarcinomas to investigate the expression of OTX1 and OTX2 genes through immunohistochemical and real-time PCR analyses.Both OTX1 and OTX2 were absent in all the samples of sinonasal Intestinal-Type Adenocarcinomas (ITACs). OTX1 mRNA was identified only in Non-Intestinal Type Adenocarcinomas (NITACs) while OTX2 mRNA was expressed only in Olfactory Neuroblastomas (ONs). We have demonstrated that the differential gene expression for both OTX1 and OTX2 genes might be a useful molecular marker to distinguish the different types of sinonasal tumors.

In the normal mucosa we observed strong and homogenous nuclear reactivity for OTX genes both in the ciliated pseudostratified respiratory-type epithelium and in the submucosal glandular cells (Figure 1A). We found nuclear expression for OTX1 in all NITACs samples (Figure 1B), while little or absent immunoreactivity was highlighted in ITACs (Figure 1C). Intense immunoreactivity was present in all ONs ...

Watch this Scientific Journal Video about Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas at JoVE.com

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas

Homeobox genes are regulatory genes often associated with tumors in the adult organisms. We investigated their comparative expression by immunohistochemical and real-time PCR analysis, in normal and inflammatory nasal mucosae and in sinonasal neoplasms in order to use them as possible diagnostic and therapeutic targets.

OTX homeobox (HB) genes are expressed during embryonic morphogenesis and during the development of olfactory epithelium in adult organisms. Mutations ...

identification-otx1-otx2-as-two-possible-molecular-markers-for

Research

JoVE Journal

Cancer Research

5.8K Views.  University of Insubria. Het algemene doel van dit experiment is om nuttige moleculaire markers voor sinonasal carcinomen en reukneuroblastomen op een eenvoudige en efficiënte manier te identificeren. Deze methode kan helpen bij het beantwoorden van belangrijke vragen op het gebied van reukneuroblastoom en sinonasal carcinoom, zoals de identificatie van moleculaire markers om tumorsubtype te discrimineren. Het belangrijkste voordeel van deze techniek is dat we in minder dan zes uur, of binnen een dag voor het zieken...

Video: Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. 
Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell1. Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)2-4 or neurodegenerative (e.g. Parkinson, Alzheimer) diseases4,5. Olfactory mucosa can be used for either comparative molecular studies4,6 or in vitro experiments on neurogenesis3,7.
The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria8-10. We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)11.
Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease4. We and others have also shown that OE-MSCs are promising candidates for cell therapy, after a spinal cord trauma12,13, a cochlear damage14 or in an animal models of Parkinson's disease15 or amnesia16.
In this study, we present methods to biopsy olfactory mucosa in rats and humans. After collection, the lamina propria is enzymatically separated from the epithelium and stem cells are purified using an enzymatic or a non-enzymatic method. Purified olfactory stem cells can then be either grown in large numbers and banked in liquid nitrogen or induced to form spheres or differentiated into neural cells. These stem cells can also be used for comparative omics (genomic, transcriptomic, epigenomic, proteomic) studies.

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain trauma/disease.

The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external ...

Neuroscience

Integration of Bioinformatics Approaches and Experimental Validations to Understand the Role of Notch Signaling in Ovarian Cancer

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to interference with proper development and may even result in initiation or progression of cancers in certain cases. Because this pathway serves complex and versatile functions, it can be studied extensively through many different approaches. Of these, bioinformatics provides an undeniably cost-efficient, approachable, and user-friendly method of study. Bioinformatics is a useful way to extract smaller pieces of information from large-scale datasets. Through the implementation of various bioinformatics approaches, researchers can quickly, reliably, and efficiently interpret these large datasets, yielding insightful applications and scientific discoveries. Here, a protocol is presented for integration of bioinformatics approaches to investigate the role of Notch signaling in ovarian cancer. Furthermore, bioinformatics findings are validated through experimentation.

Bioinformatics is a useful way to process large-scale datasets. Through the implementation of bioinformatics approaches, researchers can quickly, reliably, and efficiently obtain insightful applications and scientific discoveries. This article demonstrates the utilization of bioinformatics in ovarian cancer research. It also successfully validates bioinformatics findings through experimentation.

Notch signaling is a highly conserved regulatory pathway involved in many cellular processes. Dysregulation of this signaling pathway often leads to ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection of E6/E7 mRNA by quantitative reverse transcription polymerase chain reaction (qRT-PCR) requires RNA extraction which destroys the tumor tissue context critical for morphological correlation and has been difficult to be adopted in routine clinical practice. Our recently developed RNA in situ hybridization technology, RNAscope, permits direct visualization of RNA in formalin-fixed, paraffin-embedded (FFPE) tissue with single molecule sensitivity and single cell resolution, which enables highly sensitive and specific in situ analysis of any RNA biomarker in routine clinical specimens. The RNAscope HPV assay was designed to detect the E6/E7 mRNA of seven high-risk HPV genotypes (HPV16, 18, 31, 33, 35, 52, and 58) using a pool of genotype-specific probes. It has demonstrated excellent sensitivity and specificity against the current &#39;gold standard&#39; method of detecting E6/E7 mRNA by qRT-PCR. HPV status determined by RNAscope is strongly prognostic of clinical outcome in oropharyngeal cancer patients.

RNAscope for In situ Detection of Transcriptionally Active Human Papillomavirus in Head and Neck Squamous Cell Carcinoma

The presence of high-risk HPV in head and neck tumor tissue is associated with favorable outcomes. The recently developed RNA in situ hybridization technique called RNAscope allows direct visualization of HPV E6/E7 mRNA in FFPE tissue sections.

The &#39;gold standard&#39; for oncogenic HPV detection is the demonstration of transcriptionally active high-risk HPV in tumor tissue. However, detection ...

Medicine

Olfactory Neurons Obtained through Nasal Biopsy Combined with Laser-Capture Microdissection: A Potential Approach to Study Treatment Response in Mental Disorders

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium carbonate. Animal studies as well as human genetic studies indicate that lithium affects molecular targets that are involved in neuronal growth, survival and maturation, and notably molecules involved in Wnt signaling. Given the ethical challenge to obtaining brain biopsies for investigating dynamic molecular changes associated with lithium-response in the central nervous system (CNS), one may consider the use of neurons obtained from olfactory tissues to achieve this goal.The olfactory epithelium contains olfactory receptor neurons at different stages of development and glial-like supporting cells. This provides a unique opportunity to study dynamic changes in the CNS of patients with neuropsychiatric diseases, using olfactory tissue safely obtained from nasal biopsies. To overcome the drawback posed by substantial contamination of biopsied olfactory tissue with non-neuronal cells, a novel approach to obtain enriched neuronal cell populations was developed by combining nasal biopsies with laser-capture microdissection. In this study, a system for investigating treatment-associated dynamic molecular changes in neuronal tissue was developed and validated, using a small pilot sample of BD patients recruited for the study of the molecular mechanisms of lithium treatment response.

In this study, a novel platform to investigate intraneuronal molecular signatures of treatment response in bipolar disorder (BD) was developed and validated. Olfactory epithelium from BD patients was obtained through nasal biopsies. Then laser-capture microdissection was combined with Real Time RT PCR to investigate the molecular signature of lithium response in BD.

Bipolar disorder (BD) is a severe neuropsychiatric disorder with poorly understood pathophysiology and typically treated with the mood stabilizer, lithium ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

Pathology Assessment of PN-MPNST Progression in the P0-GGFβ3 Mouse Model

Identifying, Diagnosing, and Grading Malignant Peripheral Nerve Sheath Tumors in Genetically Engineered Mouse Models

Patients with the autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1) commonly develop plexiform neurofibromas (PNs) that subsequently transform into highly aggressive malignant peripheral nerve sheath tumors (MPNSTs). Understanding the process by which a PN transforms into an MPNST would be facilitated by the availability of genetically engineered mouse (GEM) models that accurately replicate the PN-MPNST progression seen in humans with NF1. Unfortunately, GEM models with Nf1 ablation do not fully recapitulate this process. This led us to develop P0-GGF&#946;3 mice, a GEM model in which overexpression of the Schwann cell mitogen neuregulin-1 (NRG1) in Schwann cells results in the development of PNs that progress to become MPNSTs with high frequency. However, to determine whether tumorigenesis and neoplastic progression in P0-GGF&#946;3 mice accurately model the processes seen in NF1 patients, we had to first prove that the pathology of P0-GGF&#946;3 peripheral nerve sheath tumors recapitulates the pathology of their human counterparts.
Here, we describe the specialized methodologies used to accurately diagnose and grade peripheral nervous system neoplasms in GEM models, using P0-GGF&#946;3 and P0-GGF&#946;3;Trp53+/- mice as an example. We describe the histologic, immunohistochemical, and histochemical methods used to diagnose PNs and MPNSTs, how to distinguish these neoplasms from other tumor types that mimic their pathology, and how to grade these neoplasms. We discuss the establishment of early-passage cultures from GEM MPNSTs, how to characterize these cultures using immunocytochemistry, and how to verify their tumorigenicity by establishing allografts. Collectively, these techniques characterize the pathology of PNs and MPNSTs that arise in GEM models and critically compare the pathology of these murine tumors to their human counterparts.

Author Spotlight: Genetically Engineered Mouse Models and Pathological Characterization of Neurofibromatosis Type 1 Associated Tumors

We have developed a methodology for assessing whether nervous system neoplasms in genetically engineered mice accurately recapitulate the pathology of their human counterparts. Here, we apply these histologic techniques, defined pathologic criteria, and culture methodologies to neurofibromas and malignant peripheral nerve sheath tumors arising in the P0-GGF&#946;3 mouse model.

Patients with the autosomal dominant tumor susceptibility syndrome neurofibromatosis type 1 (NF1) commonly develop plexiform neurofibromas (PNs) that ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system. They provide multiple experimental advantages, such as accessibility to numerous imaging approaches, electrophysiological techniques and behavioral assays. The Xenopus tadpole olfactory system is particularly well suited to investigate the function of synapses established during normal development or reformed after injury. Here, we describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae. We outline a combination of in vivo measurements of presynaptic calcium responses in glomeruli of the olfactory bulb with olfactory-guided behavior assays. Methods can be combined with the transection of olfactory nerves to study the rewiring of synaptic connectivity. Experiments are presented using both wild-type and genetically modified animals expressing GFP reporters in central nervous system cells. Application of the approaches described to genetically modified tadpoles can be useful for unraveling the molecular bases that define vertebrate behavior.

Functional Evaluation of Olfactory Pathways in Living Xenopus Tadpoles

Xenopus tadpoles offer a unique platform to investigate the function of the nervous system in vivo. We describe methodologies to evaluate the processing of olfactory information in living Xenopus larvae in normal rearing conditions or after injury.

Identification of OTX1 and OTX2 As Two Possible Molecular Markers for Sinonasal Carcinomas and Olfactory Neuroblastomas

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