Name: identification of otx1 and otx2 as two possible molecular markers for sinonasal carcinomas and olfactory neuroblastomas
Uploaded: 2019-02-28T13:00:00
Description: 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

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...

<ol>
	<li>Boncinelli, E., Simeone, A., Acampora, D., Gulisano, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeobox+genes+in+the+developing+central+nervous+system.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+Brain+Transcription+Factor+OTX1+Occurs+in+a+Subset+of+Normal+Germinal-Center+B+Cells+and+in+Aggressive+Non-Hodgkin+Lymphoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unsuspected+role+of+the+brain+morphogenetic+gene+Otx1+in+hematopoiesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+homeobox+OTX2+and+OTX1+in+the+early+developing+human+brain.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deregulated+homeobox+gene+expression+in+cancer:+cause+or+consequence?.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OTX1+and+OTX2+expression+correlates+with+the+clinicopathologic+classification+of+medulloblastomas.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OTX1+expression+in+breast+cancer+is+regulated+by+p53.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OTX1+promotes+colorectal+cancer+progression+through+epithelial-mesenchymal+transition.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+CRX+and+OTX2+expression+in+human+retina+and+retinoblastoma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+control+of+the+cell+cycle+in+mammary+gland+development+and+tumorigenesis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+phenotypic+analysis+of+poorly+differentiated+sinonasal+neoplasms:+an+integrated+approach+for+early+diagnosis+and+classification.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sinonasal+carcinoma:+clinical,+pathological,+genetic+and+therapeutic+advances.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> World health organization classification of tumors. Pathology and genetics of head and neck tumors. 9, (2005).</li><li>Radulesku, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endoscopic+surgery+for+sinonasal+tumors:+The+transcribriform+approach.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p53+mutations+in+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profile+of+TP53+gene+mutations+in+sinonasal+cancer.">Profile of TP53 gene mutations in sinonasal cancer.</a> Mutat Res. 686 (1-2), 9-14 (2010).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="642">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RecoverAll Total Nucleic Acid Isolation</td>
			<td style="width:140px;">Applied Biosystem</td>
			<td style="width:119px;">AM1975</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">High-Capacity cDNA Reverse Transcription Kit</td>
			<td style="width:140px;">Applied Biosystem</td>
			<td style="width:119px;">4368814</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Universal PCR Master Mix, no AmpErase UNG</td>
			<td style="width:140px;">Applied Biosystem</td>
			<td style="width:119px;">4326614</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ABI Prism 7000&#160;</td>
			<td style="width:140px;">Applied Biosystem</td>
			<td style="width:119px;">270001857</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">ACTB probe</td>
			<td style="width:140px;">Applied Biosystem</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Out of production. Sequence protected by copyright</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">OTX1 probe</td>
			<td style="width:140px;">Applied Biosystem</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Out of production. Sequence protected by copyright</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">OTX2 probe</td>
			<td style="width:140px;">Applied Biosystem</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Out of production. Sequence protected by copyright</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acqueous Hydrogen Peroxide</td>
			<td style="width:140px;">Merk</td>
			<td style="width:119px;">1072090250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Citrate Buffer</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">20276292</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triton</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">101473728</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris</td>
			<td style="width:140px;">Merk</td>
			<td style="width:119px;">108382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">NaCl</td>
			<td style="width:140px;">Merk</td>
			<td style="width:119px;">106404</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Goat Anti-OTX2 Antibody</td>
			<td style="width:140px;">Vector Laboratories</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Out of production. Catalog number not available</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rabbit Anti-Goat Antibody</td>
			<td style="width:140px;">Vector Laboratories</td>
			<td style="width:119px;">BA5000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ABC-Peroxidase Complex</td>
			<td style="width:140px;">Vector Laboratories</td>
			<td style="width:119px;">PK6100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">3,3&#39;-diaminobenzidine tetrahydrocloride (DAB)</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">D5905</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Harris Hematoxylin</td>
			<td style="width:140px;">Bioptica</td>
			<td style="width:119px;">0506004/L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pertek</td>
			<td style="width:140px;">Kaltek SRL</td>
			<td style="width:119px;">1560</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BioClear</td>
			<td style="width:140px;">Bioptica</td>
			<td style="width:119px;">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 ...

The overall goal of this experiment is to identify useful molecular markers for sinonasal carcinomas and olfactory neuroblastomas in a simple and efficient way. This method can help answer key question in the field of olfactory neuroblastoma and sinonasal carcinoma, such as the identification of molecular markers to discriminate tumor subtype. The main advantage of this technique is that we can obtain result in less than six hour, or within a day for the hospital report.Demonstrating the procedure of RNA extraction and real-time PCR will be Dr.Giorgia Millefanti, a PhD student from my lab. And demonstrating the procedure of immunohistochemistry will be Giovanni Micheloni, also a PhD student from my lab. To begin the procedure, collect and divide all the human FFPE samples into different sub-groups, according to the WHO classification of head and neck tumors.Heat the samples slides in an oven at 60 degrees Celsius for 30 minutes. Next, rehydrate the three micrometer thick FFPE sections by briefly washing them in xylene for 10 minutes. Then, wash the slides in 100%95%85%and 75%alcohol serially for five minutes each wash.Rinse the slides for five minutes in distilled water. Subsequently, block the endogenous activity by placing the slides in 3%aqueous hydrogen peroxide for 12 minutes. Afterward, perform antigen-retrieval by treating the slides with 10 millimolar citrate buffer, and microwaving for 10 minutes.After 10 minutes, wash the sections in TBS buffer. Then, add 0.2%of Triton X, and incubate them overnight at four degrees Celsius, with Goat Anti-Human OTX2 Antibody, diluted at a 1 to 100 ratio. The next day, add biotinylated rabbit anti-goat secondary antibody, diluted at 1 to 200, and incubate the sections for one hour at room temperature.Then, treat the samples with the ABC peroxidase complex. Afterward, develop the immunoreaction using diaminobenzene tetrahydrochloride, and counter stain the nuclei with Harris hematoxylin. Then, dehydrate the sections in a crescent alcohol scale, and clarify them in a clearing substance of turpine origin.Next, mount the sections on a slide with mounting medium. After RNA extraction and reverse transcription, perform quantitative real time PCR analysis with probe-based technology and a thermal cycler. Prepare the PCR reaction mix using 12.5 microliters of probe-based master mix, 1.25 microliters of each OTX1, OTX2, and ACTB probes, 50 nanograms of CDNA, and nuclease-free water up to 25 microliters of total volume.Perform all the reactions in triplicate using ACTB gene as the endogenous control to normalize gene expression levels. Next, centrifuge the plate at 1109 times gravity for three minutes. Then store the plate protected from light at four degrees Celsius, until the experiment.To perform real time PCR, place the samples in the machine. Then, set the thermal cycler profile with an initial hot start cycle at 50 degrees Celsius for two minutes, and 95 degrees Celsius for 10 minutes, followed by 40 cycles at 95 degrees Celsius for 15 seconds and a final cycle at 60 degrees Celsius for one minute. For gene expression level analysis, normalize the gene expression levels through the comparative cycle threshold method using ACTB gene as the endogenous control.Evaluate the gene expression levels using the comparative cycle threshold method, and plot the results. Then, perform statistical analysis using student's t-test, considering statistically significant results with p<0.05. In the normal mucosa, strong and homogenous nuclear reactivity for OTX genes both in ciliated pseudostratified respiratory type epithelium and in the submucosal glandular cells were observed.Nuclear expression for OTX1 was found in all non-intestinal type adenocarcinoma samples, while little or absent immunoreactivity was highlighted in intestinal type adenocarcinomas. Intense immunoreactivity was present in all olfactory neuroblastomas. Among all poorly differentiated neuro-endocrine carcinomas, OTX expression varied in intensity and percentage of positive cells.While attempting this procedure, it is important to remember to perform every single step of the RNA extraction in sterile conditions using also sterile equipment. In parallel with this procedure, other methods like x-ray analysis, endoscopic exam of nasal cavity, CT scan, magnetic resonance imaging, and biopsy, can be performed in order to confirm the results obtained from our technique. After its development, this technique paved the way for researchers in the field of oncology to explore this disease in cell models.After watching this video, you should have a good understanding of how to identify molecular markers to discriminate tumor subtypes using the immunohistochemistry and real time PCR. Don't forget that working with these reagents, instrumentation, and samples can be extremely hazardous and precautions such as DPI should always be taken when performing these procedure.

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

Research

JoVE Journal

Cancer Research

Sectioning Mammary Gland Whole Mounts for Lesion Identification

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&#38;E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&#38;E section.&#160; The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&#38;E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&#38;E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&#38;E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

We developed a method to successfully remove, process, section, and stain, for histopathological evaluation, mammary tissue that had originally been fixed on slides as whole mounts. This method may promote the collection and evaluation of mammary gland whole mounts in reproductive and developmental test guideline studies.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in several diseases including cancer. The free-living nematode C. elegans possesses an evolutionary and functionally conserved EGFR-RAS-ERK MAP signal cascade which is central for the development of the vulva. Gain of function mutations in RAS homolog LET-60 and EGFR homolog LET-23 induce the generation of visible nonfunctional ectopic pseudovulva along the ventral body wall of these worms. Previously, the multivulval (Muv) phenotype in these worms has been shown to be inhibited by small chemical molecules. Here we describe a protocol for using the worm in a liquid-based assay to identify inhibitors that abolish the activities of EGFR and RAS proteins. Using this assay, we show R-fendiline, an indirect inhibitor of K-RAS, suppresses the Muv phenotype expressed in the let-60(n1046) and let-23(sa62) mutant worms. The assay is simple, inexpensive, is not time consuming to setup, and can be used as an initial platform for the discovery of anticancer therapeutics.

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR proteins.

The changes in the plasma membrane localization of the epidermal growth factor receptor (EGFR) and its downstream effector RAS have been implicated in ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...