Name: evaluation of biomarkers in glioma by immunohistochemistry on paraffin-embedded 3d glioma neurosphere cultures
Uploaded: 2019-01-09T15:00:00
Description: Watch this Scientific Journal Video about Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures at JoVE.com

This work was supported by National Institutes of Health/National Institute of Neurological Disorders &#38; Stroke (NIH/NINDS) Grants R37-NS094804, R01-NS074387, R21-NS091555 to M.G.C.; NIH/NINDS Grants R01-NS076991, R01-NS082311, and R01-NS096756 to P.R.L.; NIH/NINDS R01-EB022563; 1UO1CA224160; the Department of Neurosurgery; Leah&#39;s Happy Hearts to M.G.C. and P.R.L.; and RNA Biomedicine Grant F046166 to M.G.C. F.M.M. is supported by an F31 NIH/NINDS-F31NS103500. J.P. was supported by a Fulbright-Argentinian Ministry of Sports and Education Fellowship.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Michigan.
1. Generation of Brain Tumor Neurospheres Derived from a Mouse Model
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In this article, we detail a versatile and reproducible method to perform immunohistochemistry on paraffin-embedded glioma NS, which maintain the characteristic 3D structure of tumor cells growing in the brain in situ. This technique possesses the following advantages over using cells plated onto glass or plastic surfaces: i) preservation of the 3D structure of NS; ii) avoiding of stress of cell dissociation before analysis of protein expression; iii) quenching of any endogenous fluorophore expression from genet...

Gliomas are primary solid tumors of the central nervous system classified by their phenotypic and genotypic characteristics according to the World Health Organization1. This classification incorporates the presence or absence of driver mutations, which represent an attractive source of biomarkers2. Biomarkers are biological characteristics that can be measured and evaluated to indicate normal and pathological processes, as well as pharmacological responses to a therapeutic intervention3. Biomarkers can be detected in tumor tissue and cells derived from glioma, enabling its biological characterization in different aspects. Some examples of glioma biomarkers include mutated isocitrate dehydrogenase 1 (IDH1), a mutant enzyme characteristic of lower-grade gliomas associated with better prognosis4. Mutated IDH1 is frequently expressed in combination with TP53 and alpha-thalassemia/mental retardation syndrome X-linked (ATRX)-inactivating mutations, defining a specific glioma subtype4,5. ATRX-inactivating mutations also occur in 44% of pediatric high-grade gliomas2,6 and have been associated with aggressive tumors and genomic instability6,7. In addition, pediatric diffuse intrinsic pontine gliomas (DIPG) are differentiated in subgroups according to the presence or absence of a K27M mutation in histone H3 gene H3F3A, or in the HIST1H3B/C gene1,6. Therefore, the introduction of molecular characterization permits the subdivision, study, and treatment of gliomas as separate entities, which will more allow the development of more targeted therapies for each subtype. In addition, the analysis of biomarkers can be used to evaluate different biological processes such as apoptosis8, autophagy9, cell cycle progression10, cell proliferation11, and cell differentiation12.
Genetically engineered animal models that harbor the genetic lesions present in human cancers are critical for the study of signaling pathways that mediate disease progression. Our laboratory has implemented the use of the Sleeping Beauty (SB) transposase system to develop genetically engineered mouse models of glioma harboring specific mutations that recapitulate human glioma subtypes13,14. These genetically engineered mouse models are used for generating tumor-derived NS, which enable in vitro studies in a 3D system, mirroring salient features present in the tumors in situ15. Also, the orthotopical transplantation of NS into mice can generate secondary tumors that retain features of the primary tumor and mimic the histopathological, genomic, and phenotypic properties of the corresponding primary tumor15.
In serum-free culture, brain tumor cells with stem cell properties can be enriched, and in the presence of epidermal growth factors (EGF) and fibroblast growth factors (FGF), they can be grown as single cell-derived colonies such as NS cultures. In this selective culture system, most differentiating or differentiated cells rapidly die, whereas stem cells divide and form the cellular clusters. This allows the generation of a NS culture that maintains glioma tumor features16,17,18. Glioma NS can be used to evaluate several aspects of tumor biology, including the analysis of biomarkers that have applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. Here, we detail a protocol to generate glioma NS and embed the 3D cultures in paraffin to be used for IHC staining. One advantage of fixing and embedding glioma NS is that the morphology of NS is maintained better compared to the conventional cytospin method19, in which glioma NS are subjected to stressful manipulation for cell dissociation and become flattened. In addition, embedding quenches any endogenous fluorophore expression from genetically engineered tumor NS, enabling staining across the fluorescent spectra. The overall goal of this method is to preserve the 3D structure of glioma cells through a paraffin embedding process and enable characterization of glioma neurospheres using immunohistochemistry.

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			<td height="21" style="height:21px;width:305px;">Coated microtome blade HP35</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:221px;">3150734</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microtome RM 2135</td>
			<td style="width:191px;">Leica</td>
			<td style="width:221px;">MR2135</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Formalin solution, neutral buffered, 10%</td>
			<td style="width:191px;">Sigma-aldrich</td>
			<td>HT501128-4L</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:305px;">Rabbit polyclonal anti-ATRX,</td>
			<td style="width:191px;">Santa Cruz Biotechnology</td>
			<td style="width:221px;">sc-15408</td>
			<td>IHC, 1:250 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Rabbit polyclonal anti-Ki-67</td>
			<td style="width:191px;">Abcam</td>
			<td style="width:221px;">Ab15580</td>
			<td>IHC, 1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rabbit polyclonal anti-OLIG2</td>
			<td>Millipore</td>
			<td>AB9610</td>
			<td>IHC, 1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat polyclonal anti-rabbit biotin-conjugated</td>
			<td style="width:191px;">Dako</td>
			<td style="width:221px;">E0432</td>
			<td>IHC, 1:1000 dilution</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:305px;">Vectastain Elite ABC HRP kit</td>
			<td style="width:191px;">Vector Laboratories Inc</td>
			<td style="width:221px;">PK-6100</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">BETAZOID DAB CHROMOGEN KIT</td>
			<td style="width:191px;">Biocare medical</td>
			<td style="width:221px;">BDB2004 L/price till 12/18</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">N-2 Supplement</td>
			<td style="width:191px;">ThermoFisher</td>
			<td style="width:221px;">17502048</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">N-27 Supplement</td>
			<td style="width:191px;">ThermoFisher</td>
			<td style="width:221px;">A3582801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Accutase&#174; Cell Detachment Solution</td>
			<td style="width:191px;">Biolegend</td>
			<td style="width:221px;">423201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">AGAROSE LE</td>
			<td style="width:191px;">GoldBio</td>
			<td style="width:221px;">A-201-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Genesee Sc. Corporation</td>
			<td style="width:191px;">Olympus 15 ml</td>
			<td style="width:221px;">21-103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Genesee Sc. Corporation</td>
			<td style="width:191px;">TC-75 treated Flask</td>
			<td style="width:221px;">25-209</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">Genesee Sc. Corporation</td>
			<td style="width:191px;">TC-25 treated Flask</td>
			<td style="width:221px;">25-207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">DMEM/F12</td>
			<td style="width:191px;">Gibco</td>
			<td style="width:221px;">11330-057</td>
			<td>NS media</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:305px;">HBSS</td>
			<td style="width:191px;">GibcoTM</td>
			<td style="width:221px;">14175-103</td>
			<td>balanced salt solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">C57BL/6</td>
			<td style="width:191px;">Taconic</td>
			<td style="width:221px;">B6-f</td>
			<td>C57BL/6 mouse</td>
		</tr>
	</tbody>
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evaluation of biomarkers in glioma by immunohistochemistry on paraffin-embedded 3d glioma neurosphere cultures

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as biomarkers with applications in diagnosis, prognosis, classification, state of tumor progression, and cell differentiation state. These analyses of biomarkers are also useful to characterize tumor neurospheres (NS) generated from glioma patients and glioma models. Tumor NS provide a valuable in vitro model to assess different features of the tumor from which they are derived and can more accurately mirror glioma biology. Here we describe a detailed method to analyze biomarkers in tumor NS using immunohistochemistry (IHC) on paraffin-embedded tumor NS.

To monitor tumor development and evaluate if the tumor has reached the size necessary to generate NS, we analyzed the luminescence emitted by tumors using bioluminescence. This allows the study of transduction efficiency in the pups and tumor progression by the luminescence signal of luciferase (Figure 1A and 1B). When the tumor size reaches a signal of 1 x 107 photons/s/cm2/sr (Figure 1</stron...

Watch this Scientific Journal Video about Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures at JoVE.com

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural differentiation.

Analysis of protein expression in glioma is relevant for several aspects in the study of its pathology. Numerous proteins have been described as ...

This protocol is significant because can be used to study the presence of glioma biomarker by a method that preserves the 3D structure of tumor neurospheres. The main advantage of this protocol is that the morphology of neurospheres is maintained compared to cytospin method in which cells are subjected to stressful manipulation and become flattened. This method can be applied to any other tissue culture systems in which cells are cultured in 3D and maintenance of the structure is important.To begin, culture tumor neurospheres or NS in a T-25 flask until the cells are confluent. Elect the tumor NS in a 15 milliliter conical tube. Impellet the NS at 300 Gs for five minutes.After this, re-suspend the pellet in one milliliter of 10%formalin and incubate the tube at room temperature for 10 minutes. Add five milliliters of HBSS to the re-suspended NS.Impellet the cells as previously described. Place the tube containing the pellet on ice.Re-suspend the washed NS pellet in 500 microliters of two percent warm liquid agarose. And mix by a gentle pipetting or stirring. Place the agarose embedded NS on ice until the agarose polymerizes.Then remove the piece of agarose, holding the NS.Place the piece of agarose into a cassette for tissue processing. Set a water bath to 42 degrees Celsius. And carefully insert the blade into the microtome.Then place the paraffin block into the microtome. With one hand, press down on the lever that controls the thickness of each section located on the left hand side of the machine. Press the lever to the second stop to set the microtome to a 30 micrometer thickness.Turn the coarse hand wheel to begin trimming the block. Once the surface of the sample is almost exposed, set the lever that controls thickness to five or 10 micrometers. Stop trimming when the surface of the sample is reached.Fill an ice box with ice and just enough water to protect the paraffin block from directly touching the ice. Discard the old microtome blade and insert a new one for sectioning. Dry the paraffin block with gauze and place it on the microtome.Turn the coarse hand wheel to begin sectioning the paraffin. Then use a damp paintbrush to transfer the paraffin ribbon to the 42 degree Celsius water bath. Using the curve of the forceps, place the forceps in between two paraffin sections and push the sections apart gently.Use the damp paintbrush to push the separated sections onto positively charged adhesion microscope slides. First, melt the paraffin by incubating the slides in a metal rack at 60 degrees Celsius for 20 minutes. Then deparaffinize the slides via incubation in a re-hydration train at room temperature according to the text protocol.Store the slides in tap water to prevent non-specific antibody binding. After this, incubate the slides in citrate buffer at 125 degrees Celsius for 30 seconds and 90 degrees Celsius for 10 seconds. Allow the slides to cool before rinsing them five times with distilled water.Next, incubate the slides at room temperature in 0.3%hydrogen peroxide for 20 minutes. Then, wash the slides for five minutes, three times in washing buffer with gentle agitation. Incubate the slides overnight in a humidified chamber set to four degrees Celsius with diluted primary antibody.After this, wash the slides three times for five minutes with gentle agitation. Next, incubate the slides in diluted biotinylated secondary antibody at room temperature for an hour. Wash the slides three times for five minutes in washing buffer with gentle agitation.Then, incubate the slides in avidin-biotinylated horseradish peroxidase complex at room temperature in the dark for an hour. Repeat the wash as previously described. After this, incubate the slides with DAB hydrogen peroxide substrate brands for two to five minutes at room temperature.Rinse the slides with tap water and dip the slides in hematoxylin solution to counterstain them. Then rinse the slides thoroughly in tap water until the water runs clear. Dehydrate the slides according to the text protocol.Finally, coverslip the slides with a xylene-based mounting medium and allow them dry on a flat surface at room temperature. In this protocol, NS are fixed and embedded in agarose and paraffin. And then they are stained for specific glioma biomarkers.The expression level of ATRX, a chaperone protein that mediates chromatin structure, is low. Ki67, a biomarker of cell proliferation, is positive in the glioma NS.Finally, the NS are also positive for OLIG2, a transcription factor that mediates oligodendrocyte differentiation. The most important thing to remember is to re-suspend the cells well in the warm agarose before transferring to ice, to ensure an even distribution of neurospheres.

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