This work was supported by the National Institute of Deafness and Other Communication Disorders grants R01DC015824 (K.M.S.) and T32DC00038 (supporting J.E.S and S.D.), the Department of Defense grant W81XWH-14-1-0091 (K.M.S.), the Bertarelli Foundation (K.M.S.), the Nancy Sayles Day Foundation (K.M.S.), the Lauer Tinnitus Research Center (K.M.S.), and the Barnes Foundation (K.M.S.).

<ol>
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The authors have nothing to disclose.

This manuscript describes a unified methodological framework for VS research, outlining the simultaneous processing of human VS and GAN specimens for downstream research applications. As VS research enters the age of precision medicine, preparing the same sample in forms capable of answering numerous research questions will enable the discovery of molecular, cellular, genetic, and proteomic insights specific to individual patients.
The purity of human Schwann cell and VS cultures were thoroughly assessed over time by means of immunocytochemistry, using the ratio of cells positive for the S100 marker to those positive for the DAPI nuclear stain19. Accordingly, it is recommended to perform experiments on primary human Schwann cell cultures within the first two weeks after plating the cells, as the purity of these cells is the highest during this period; afterwards, fibroblast-like cells begin to predominate. Though VS cell cultures are also maximally pure at two weeks in culture, VS cells lack contact-mediated inhibition and will continue to proliferate at later stages. Additionally, a direct microarray comparison of protein expression from VS tissue (section 4) and VS cells in culture from the same tumors (section 5) successfully showed that VS cultures demonstrate a satisfactorily high level of biological similarity to their respective parent tumors19. The successful quantification of proteins of interest can be performed via Western blot of protein lysates generated in section 4 and the quantification of RNA transcripts via qRT-PCR of RNA extracted from tissues preserved with RNA stabilization solution (section 2)12,13,14.
VS and GAN cells in culture can be exposed to chemically active substances, such as nonsteroidal anti-inflammatory drugs, small interfering RNAs (siRNAs), or natural organic compounds to elucidate disease-specific molecular mechanisms or to test a therapeutic target13,14,29. Compounds of interest can be directly added to cells in culture after appropriate dilutions in regular supplemented culture medium. To assess the effect of such substances on important aspects of cellular physiology, bromodeoxyuridine (BrdU) labeling for proliferation, terminal deoxynucleotidyl tranferase dUTP nick end labeling (TUNEL) for apoptosis, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction for metabolic viability are reliable assays that can be used with confidence on these cells19. Culture medium from treated cells can also be carefully aspirated, stored at -20 &#176;C, and tested to quantify relative levels of secreted substances, such as prostaglandin E2, the secretion of which can be affected by drug treatment13. Additionally, tissue fixation (section 7) and subsequent embedding in OCT or paraffin provides a robust substrate for immunofluorescence assays13,14.
The tumor- or nerve-conditioned medium generated in section 3 provides a simple and effective way to assess VS-secreted soluble molecules and to extract extracellular vesicles. Cytokine arrays or ELISAs can be carried out on these secretions, as indicated in the manufacturers&#39; protocols, outlined in two published studies9,12. Extracellular vesicles can be purified from these secretions using ultracentrifugation, as detailed in a previous publication30. Alternatively, the secretions themselves can be used as patient-specific reagents for downstream research applications. For example, in an experiment that revealed a novel mechanism underlying VS-associated sensorineural hearing loss, secretions were collected from the VS tissue of patients with poor hearing and VS patients with good hearing after incubation in DMEM for 72 h (section 3). These tumor secretions were then applied to murine cochlear explants, and it was revealed that tumor secretions from VS patients with poor hearing caused more damage to cochlear explants than secretions from VS patients with good hearing15. Importantly, secretions collected at time points before 72 h did not result in substantial damage.
Human perilymph collected from patients undergoing translabyrinthine VS resection (section 8) represents an exciting new avenue for the discovery of VS biomarkers. Specimens collected according to section 8 were analyzed via liquid chromatography-tandem mass-spectrometry (LC-MS/MS) to map the proteome of human perilymph, and 15 candidate biomarkers of VS were successfully identified31. A similar analysis could be possible using human CSF collected from VS patients (section 9).
A significant consideration in VS research is the selection of appropriate control tissue. Previous studies have used the cochlear nerve, the vestibular nerve, and unrelated peripheral nerves (such as the sciatic nerve), to variable effect22,32. However, several reasons lead to the support of the use of GANs as maximally relevant controls15. Similar to the vestibular nerve, the GAN is a peripheral, sensory nerve with axons ensheathed by Schwann cells. Importantly, a literature search reveals that schwannomas have never been found to develop from the GAN, making neoplastic changes in this tissue improbable. Additionally, GANs are regularly sacrificed when accessing structures of the deeper neck, giving hospital-based researchers easy access to healthy specimens during routine neck dissections and parotidectomies. Although the cochlear nerve is often sacrificed during VS surgery and may be readily available, its close proximity to the vestibular nerves risks the sharing of the same microenvironment, which may demonstrate pre-tumorous molecular changes33. Other laboratories have also successfully utilized GANs as an adequate control for VS research, and it is recommended to continue this practice20,21,22,34.
One critical but often overlooked step within the cell type-specific culture protocols (sections 5 and 6) is the proper removal of non-viable tissue and blood vessels. Removal of these non-tumor tissues is crucial to generate robust cultures of maximal purity. Furthermore, when plating cell-containing culture medium onto coverslips, it is important to pay close attention to the amount of tissue transferred to each well. Achieving an even, mildly dense distribution of cells containing a few &#34;chunks&#34; of denser tissue in each well is particularly important for Schwann cells, which require a sufficient number of adherent cells to thrive. Coating the coverslips with poly-D-lysine and laminin also allows for the efficient growth of cells. Cells proliferate more robustly when the coverslips are coated in the laboratory, as compared to commercially available pre-coated products.
To ensure a high-quality protein and RNA extraction, verify that the tissue remains at temperatures below 4 &#176;C throughout sections 2 and 4. Additionally, since the literature regarding the analysis of VS secretions is scarce (section 3), new projects might require further innovations and different lengths of incubation.
As methods to address VS pathobiology continue to advance, the use of this streamlined combination of fundamental techniques will allow the maximum amount of information to be gleaned from a single human sample. The informed simultaneous processing of VS and GAN tissues will prove integral to the generation of effective pharmacological and genetic therapies against this debilitating tumor.

Vestibular schwannomas (VSs) are the most common neoplasms of the cerebellopontine angle, diagnosed in 1 in every 100,000 individuals. Though non-metastatic, these tumors severely affect a patient&#39;s quality of life1,2,3,4,5,6. Affected individuals commonly live with hearing loss, tinnitus, and a feeling of aural fullness. Symptoms become increasingly debilitating as the tumor grows, causing balance problems, facial paralysis, and impairment of other cranial nerve functions. Life-threatening complications due to brainstem compression may also ensue7.
Management options for VS are essentially limited to watchful waiting for static tumors and stereotactic radiation therapy or surgical resection for growing tumors8. The surgical removal of these tumors in research-affiliated hospitals presents the opportunity to acquire and analyze fresh tumor tissue collected during patient surgeries. One specific surgical approach to VS, the translabyrinthine resection, can even offer access to valuable human sensory epithelium from the inner ear and perilymph.
Because VSs arise from a peripheral sensory nerve (i.e., the vestibular nerve), it is important to compare VS-associated observations with those derived from an appropriate control nerve, such as the human great auricular nerve (GAN). Healthy GANs are regularly sacrificed during parotidectomies or neck dissections and can be used as robust models for healthy Schwann cell physiology9.
Because there are no FDA-approved drugs for the treatment or prevention of sporadic VS, it is imperative that researchers elucidate the underlying molecular mechanisms of the disease to identify therapeutic targets. Proteins that have been shown to play a role in VS pathogenesis include merlin, vascular endothelial growth factor (VEGF), cyclooxygenase 2 (COX-2), nuclear factor kappa B (NF-&#954;B), tumor necrosis factor alpha (TNF-alpha), epidermal growth factor receptor (EGFR), and related signaling molecules10,11,12,13,14,15,16,17.
Recent advances have expanded and improved protocols for the collection, processing, culture, and downstream investigation of primary human vestibular schwannomas and healthy nerve tissues18,19. However, most existing protocols are designed to accommodate the preparation of such tissues for a single downstream research application (i.e., cell culture alone). This article presents a unified methodological framework for the simultaneous processing of a single primary human VS or GAN sample for multiple downstream applications: cell type-specific culture, protein extraction, RNA preservation, tumor secretion collection, and tissue fixation. This work also details the surgical collection and processing of human cerebrospinal fluid (CSF) and perilymph during translabyrinthine VS resection, as these closely related tissues may serve as important sources of biomarkers for VS. Finally, this protocol presents steps for the viral transduction of primary human VS cells in culture as a novel application of this tissue for use in gene therapy.

<table border="0" cellpadding="0" cellspacing="0" width="1419">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">BioCoat Poly-D-Lysine/Laminin 12mm #1 German Glass Coverslip</td>
			<td style="width:159px;">Corning</td>
			<td style="width:159px;">354087</td>
			<td style="width:683px;">Or prepare coverslips with Corning Laminin (CB-40232) and Cultrex Poly-L-Lysine (3438-100-01)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">CELLSTAR 15 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile</td>
			<td style="width:159px;">Greiner Bio-One</td>
			<td style="width:159px;">188161</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">CELLSTAR 50 ml Centrifuge Tubes, Conical bottom, Graduation, Sterile</td>
			<td style="width:159px;">Greiner Bio-One</td>
			<td style="width:159px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">CELLSTAR Cell Culture Dish, 60 mm</td>
			<td style="width:159px;">Greiner Bio-One</td>
			<td style="width:159px;">628160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Collagenase from Clostridium histolyticum, Sterile-filtered</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:159px;">C1639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Costar 24 Well Clear TC-Treated Multiple Well Plates, Sterile</td>
			<td style="width:159px;">Corning</td>
			<td style="width:159px;">3526</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">D1306</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">DMEM, high glucose, pyruvate, no glutamine, 500 ml</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">10313-039</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">DMEM/F-12, 500 ml</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">11320-033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Dumont #3 Forceps, Dumoxel</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:159px;">11231-30</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Dumont #5 Forceps, Standard tip, Inox</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:159px;">11251-20</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">Fetal Bovine Serum, qualified, USDA-approved regions, 500 ml</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">10437-028&#160;</td>
			<td>Aliquot in 50 ml tubes and store in -20&#176;C freezer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">Hyaluronidase from Bovine Testes, Type I-S, Lyophilized Powder</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:159px;">H3506</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">Millex-GP Syringe Filter Unit, 0.22 &#181;m, polyethersulfone, 33 mm, sterile</td>
			<td style="width:159px;">EMD Millipore</td>
			<td style="width:159px;">SLGP033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Paraformaldehyde, Reagent Grade, Crystalline</td>
			<td style="width:159px;">Sigma-Aldrich</td>
			<td style="width:159px;">P6148</td>
			<td>Prior to use: Establish Standard Operating Procedures based on protocols available online</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">PBS, pH 7.4, 500 ml</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">10010-023&#160;</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">Penicillin-Streptomycin (10,000 U/ml), 100 ml</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">PhosSTOP Phosphatase Inhibitor Tablets</td>
			<td style="width:159px;">Roche</td>
			<td style="width:159px;">04906845001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">Pierce Protease Inhibitor Tablets</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">88666</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Pipettes and pipette tips, 5/10/25 ml</td>
			<td style="width:159px;">Variable</td>
			<td style="width:159px;">Variable</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Plastic Homogenization Pestle for 1.5/2.0ml Microtubes</td>
			<td style="width:159px;">E&#38;K Scientific</td>
			<td style="width:159px;">EK-10539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">PrecisionGlide Needles, 27 G x 1 1/2 in&#160;</td>
			<td style="width:159px;">BD</td>
			<td style="width:159px;">301629</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">RIPA Buffer</td>
			<td style="width:159px;">Boston BioProducts</td>
			<td style="width:159px;">BP-115</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">RNAlater (RNA stabilization solution)</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:159px;">AM7021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Safe-Lock Microcentrifuge Tubes, Polypropylene, 0.5 ml</td>
			<td style="width:159px;">Eppendorf</td>
			<td style="width:159px;">022363719</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Safe-Lock Microcentrifuge Tubes, Polypropylene, 1.5 ml</td>
			<td style="width:159px;">Eppendorf</td>
			<td style="width:159px;">022363204</td>
			<td>Autoclave prior to use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Saline - 0.9% Sodium Chloride Injection, bacteriostatic, 20 ml</td>
			<td style="width:159px;">Hospira</td>
			<td style="width:159px;">0409-1966-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Scalpel Blades - #15</td>
			<td style="width:159px;">Fine Science Tools</td>
			<td style="width:159px;">10015-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Schuknecht Suction Tube 24 gauge</td>
			<td style="width:159px;">Bausch + Lomb</td>
			<td style="width:159px;">N1698 42</td>
			<td>Useful for the surgical approach (in addition to common otologic surgical instruments) and e.g. a blue surgical marker</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Specimen Container, OR sterile, 4OZ&#160;</td>
			<td style="width:159px;">Medline</td>
			<td style="width:159px;">DYND30331H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Stemi 2000-C Stereo Microscope</td>
			<td style="width:159px;">Zeiss</td>
			<td style="width:159px;">000000-1106-133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Syringe/Needle Combination, Luer-Lok Tip, 5 ml, 22 G x 1 in.</td>
			<td style="width:159px;">BD</td>
			<td style="width:159px;">309630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Tuberculin Syringe Only, Slip tip, 1 ml</td>
			<td style="width:159px;">BD</td>
			<td style="width:159px;">309659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Tuberculin Syringe Only, Slip tip, 3 ml</td>
			<td style="width:159px;">BD</td>
			<td style="width:159px;">309656</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Ultrasonic homogenizer, 4710 Series, CV18 probe</td>
			<td style="width:159px;">Cole-Parmer</td>
			<td style="width:159px;">CP25013</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 tumors cause sensorineural hearing loss in up to 95% of affected individuals, the molecular mechanisms underlying this hearing loss remain elusive. This article outlines the steps established in our laboratory to facilitate the collection and processing of various primary human tissue samples for downstream research applications integral to the study of vestibular schwannomas. Specifically, this work describes a unified methodological framework for the collection, processing, and culture of Schwann and schwannoma cells from surgical samples. This is integrated with parallel processing steps now considered essential for current research: the collection of tumor and nerve secretions, the preservation of RNA and the extraction of protein from collected tissues, the fixation of tissue for the preparation of sections, and the exposure of primary human cells to adeno-associated viruses for application to gene therapy. Additionally, this work highlights the translabyrinthine surgical approach to collect this tumor as a unique opportunity to obtain human sensory epithelium from the inner ear and perilymph. Tips to improve experimental quality are provided and common pitfalls highlighted.

Primary human VS cells in culture, as established in section 5, can be treated as informative models for disease-associated processes in many downstream research applications (Figure 1). Healthy Schwann cells cultured in section 6 provide a direct and instructive point of comparison. As outlined below, extensive data from VSs and GANs processed according to this unified methodological framework are available in multiple articles previously published12,13,14,15,27.
The successful transduction of primary VS cells with adeno-associated viruses for gene therapy is presented here for the first time (Figure 2). Primary VS cells were transduced in culture with GFP-expressing Anc80, a predicted ancestor of adeno-associated viruses28. Anc80 has been shown to be a maximally efficient vector for gene transfer in murine cochlear tissues in vitro and in vivo27. The effective transduction of primary human cells with this vector carries important implications for future forays into gene therapy for VS.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55827/55827fig1.jpg" /> 
Figure 1: Bright-field Images of Primary Human Vestibular Schwannoma Cultures. 
Typical patterns in the organization of VS cells in culture can be identified. Scale bar = 200 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55827/55827fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55827/55827fig2.jpg" /> 
Figure 2: Representative Immunofluorescent Image of Primary Human Vestibular Schwannoma Cultures after Exposure to GFP-expressing Anc80 for 48 h at a Multiplicity of Infection (MOI) of 250,000. 
Green = GFP; red = phalloidin/F-actin (stains the cytoskeleton); blue = DAPI (stains the nucleus). Scale bars = 50 &#181;m (A) and 100 &#181;m (B). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55827/55827fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Unified Methodological Framework for Vestibular Schwannoma Research at JoVE.com

A Unified Methodological Framework for Vestibular Schwannoma Research

The goal of this protocol is to outline the collection and processing of human surgical samples for multiple downstream applications in vestibular schwannoma and Schwann cell research.

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

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Cancer Research

7.3K Views.  Massachusetts Eye and Ear. The goal of this protocol is to outline the collection and processing of human surgical samples for multiple downstream applications in vestibular schwannoma and Schwann cell research.

Video: A Unified Methodological Framework for Vestibular Schwannoma Research

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

E-Patient Counseling Trial (E-PACO): Computer Based Education versus Nurse Counseling for Patients to Prepare for Colonoscopy

Improving patient education focusing on bowel preparation before a colonoscopy leads to cleaner colons. Endoscopy units must obtain informed consent and perform a risk assessment for sedative use prior to a colonoscopy. The current practice in the Netherlands to achieve these goals is nurse counseling in an outpatient setting. This is costly and has disadvantages in terms of uniformity and time consumption for both the patient and the hospital. The hypothesis is that computer-based education with use of video and 3D animations may replace nurse counseling in most cases, without losing quality of bowel cleanliness during colonoscopy.
This multicenter, randomized, endoscopist blinded clinical trial evaluates a primary outcome measure (bowel preparation) during colonoscopy. Secondary outcome measures are sickness absence, patient anxiety after instruction and prior to colonoscopy, patient satisfaction and information re-call. The study will be performed in four endoscopy units of different levels (rural, urban, and tertiary). Inclusion criteria are adult age and referral for complete colonoscopy. Exclusion criteria are Dutch illiteracy, audiovisual handicaps or mental disabilities and no (peers with) internet access.
This trial aims to establish online computer-based education as tool for patient education prior to a colonoscopy. By choosing a direct comparison with the standard of care (nurse counseling), both endoscopic quality measures and patient related outcome measures can be evaluated.

E-Patient Counseling Trial E-PACO: Computer Based Education versus Nurse Counseling for Patients to Prepare for Colonoscopy

The aim of this trial is to establish the position of online computer-based education as a tool for patient preparation prior to a colonoscopy. Computer based education is compared with the standard of care, nurse counseling, evaluating endoscopic quality measures and patient related outcome measures.

Improving patient education focusing on bowel preparation before a colonoscopy leads to cleaner colons. Endoscopy units must obtain informed consent and ...

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. Although heterogeneous in presentation, a common denominator among these tumors is the overexpression of somatostatin receptors. 68Ga-DOTATATE is a somatostatin analog labeled with the positron emitter gallium-68 (68Ga). For well-differentiated neuroendocrine tumors, 68Ga-DOTATATE positron emission tomography (PET)/computed tomography (CT) imaging is used for diagnosis, determination of disease burden, and therapy selection.
This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiolabeling of 68Ga-DOTATATE is performed with a fully automated labeling module coupled to a germanium-68 (68Ge)/68Ga generator. Quality control of the final product evaluates radiochemical purity with instant thin-layer chromatography and solid-phase chromatography, and pH prior to patient injection. Periodic quality control is performed to determine 68Ge breakthrough, sterility, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) content. Patient preparation includes patient instructions, a protocol for 68Ga-DOTATATE during treatment with somatostatin analogs, and intravenous administration of the radiopharmaceutical. For PET/CT imaging, the acquisition and reconstruction settings are described. For each step, radiation safety will be highlighted, as well as time constrictions due to the short half-life of 68Ga.
Fully automated in-house production and quality control of 68Ga-DOTATATE leads to very high success rates (95%) and produces two to four patient dosages per batch, depending on the yield of the generator. In conclusion, 68Ga-DOTATATE PET/CT imaging is a noninvasive and fast method of providing information on the tumor burden of neuroendocrine tumors (NETs) while also assisting in diagnosis and therapy selection.

A Practical Guide for the Production and PET/CT Imaging of 68Ga-DOTATATE for Neuroendocrine Tumors in Daily Clinical Practice

Well-differentiated neuroendocrine tumors overexpress somatostatin receptors which can be utilized for diagnostic imaging with the radiolabeled somatostatin analog 68Ga-DOTATATE. This protocol details the radiolabeling of 68Ga-DOTATATE, quality control, patient preparation, and subsequent PET/CT imaging. Radiation safety and time constrictions due to the short half-life of 68Ga are taken into account.

Neuroendocrine tumors are a rare form of cancer that arise from neuroendocrine cells and can be present at almost any location throughout the body. ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Monitoring eIF4F Assembly by Measuring eIF4E-eIF4G Interaction in Live Cells

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic activation in humans. eIF4F is a cap-binding complex involved in the mRNA-ribosome recruitment phase of translation initiation. In many cellular and pre-clinical model of cancers, the deregulation of eIF4F leads to increased translation of specific mRNA subsets that are involved in cancer proliferation and survival. eIF4F is a hetero-trimeric complex built from the cap-binding subunit eIF4E, the helicase eIF4A and the scaffolding subunit eIF4G. Critical for the assembly of active eIF4F complexes is the protein-protein interaction between eIF4E and eIF4G proteins. In this article, we describe a protocol to measure eIF4F assembly that monitors the status of eIF4E-eIF4G interaction in live cells. The eIF4e:4G cell-based protein-protein interaction assay also allows drug induced changes in eIF4F complex integrity to be accurately and reliably assessed. We envision that this method can be applied for verifying the activity of commercially available compounds or for further screening of novel compounds or modalities that efficiently disrupt formation of eIF4F complex.

Here, we present a protocol to measure eIF4E-eIF4G interaction in live cells that would enable the user to evaluate drug induced perturbation of eIF4F complex dynamics in screening formats.

Formation of the eIF4F complex has been shown to be the key downstream node for the convergence of signalling pathways that often undergo&#160;oncogenic ...

A 3D Spheroid Model for Glioblastoma

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were developed. These models may be particularly important in the field of neuro-oncology. Indeed, brain tumors have the tendency to invade the healthy brain environment. We describe herein an ideal 3D glioblastoma spheroid-based assay that we developed to study tumor invasion. We provide all technical details and analytical tools to successfully perform this assay.

Here, we describe an easy-to-use invasion assay for glioblastoma. This assay is suitable for glioblastoma stem-like cells. A Fiji macro for easy quantification of invasion, migration, and proliferation is also described.

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...