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
<ol>
	<li>Before the dissection procedure, prepare neural stem cell media (NSC Media: DMEM/F12 with 1x B27 supplement, 1x N2 supplement, 1x normocin, and 1x antibiotic/antimycotic supplemented with human recombinant EGF and basic-FGF at concentrations of 20 ng/mL each). Fill a 1.5 mL tube with 300 &#181;L of NSC media and reserve for later.</li>
	<li>Select a mouse with a large tumor. 
	NOTE: The size of a tumor can be monitored by bioluminescence if the plasmid or virus injected encodes luciferin. Usually, a large tumor has a bioluminescence of &#62; 106 photons/s/cm2/sr.</li>
	<li>Euthanize the mouse with an overdose of isoflurane: infuse a tissue paper with isoflurane and introduce the tissue in a euthanization chamber, avoiding direct contact with the mice to prevent irritation. Wait until the mice do not show a pedal reflex (usually 5-10 min). Decapitate the mouse and dissect the brain as described in Calinescu et al.13.</li>
	<li>If the tumors are fluorescent, use a dissecting microscope equipped with a fluorescent lamp to identify the tumor mass within the brain. With fine forceps, dissect the tumor (usually 5-7 mm in diameter) from the normal brain tissue.</li>
	<li>Place the dissected tumor in the 1.5 mL tube with NSC media (step 1.1). Homogenize the tumor with a disposable plastic pestle that fits into the walls of the microcentrifuge tube by applying gentle pressure.</li>
	<li>To dissociate the cell clumps, add 1 mL of enzyme-free dissociation media and incubate for 5 min at 37 &#176;C.</li>
	<li>Filter the cell suspension through a 70 &#181;m cell strainer and wash the strainer with 20 mL of NSC media.</li>
	<li>Centrifuge at 300 x g for 5 min, decant the supernatant, and re-suspend the pellet into 6 mL or 15 mL of NSC media, depending on the size of the pellet.</li>
	<li>Plate the tumor cell suspension into a T-25 or T-75 culture flask and culture at 37 &#176;C in a tissue culture incubator with an atmosphere of 95% air and 5% CO2.</li>
	<li>After 3 days, transfer the healthy suspended NS and re-plate them into a T-25 or T-75 tissue culture flask. If necessary, add more NSC media to the culture. 
	NOTE: There will usually be a mixed population of cells, some will be dead, some will have adhered, and some will be forming NS that will be floating in the media</li>
</ol>
2. Maintaining and Passaging the NS
NOTE: Prevent overgrowth and maintain cells at relatively high density. Confluence is determined by spheres size (black centers of large spheres mean overgrowth). The growth rate of the NS will depend on the oncogenes used to generate tumors.
<ol>
	<li>Once the NS culture has reached confluency, collect the culture in a sterile centrifuge tube and pellet the NS at 300 x g for 5 min.</li>
	<li>Discard the supernatant, add 1 mL of the cell detachment solution, and pipette to re-suspend the pellet.</li>
	<li>Incubate at 37 &#176;C for 5 min, flicking the tube every 2 min to help cell dissociation. 
	NOTE: When NS reach confluency, they can be usually seen macroscopically as small white spheres. To assure cell dissociation, at least all the visible NS must have disappeared.</li>
	<li>Add 5 mL of HBSS 1x and pipette several times. Pellet cells at 300 x g for 5 min. Discard the supernatant. Re-suspend the pellet in 5 mL of NSC media supplemented with recombinant EGF and basic-FGF.</li>
	<li>Add 1 mL of cells to 15 mL of NSC media and culture in a T-75 flask&#160;at 37 &#176;C in a tissue culture incubator with an atmosphere of 95% air and 5% CO2.</li>
	<li>Add growth factors (EGF and basic-FGF) every 3 days. If the media turns light orange and the cells are not yet confluent, change the media. To do this, centrifuge the NS at 300 x g for 4 min and re-suspend in NSC media supplemented with growth factors. 
	NOTE: Depending on the size of the pellet formed, cells may need to be split into more flasks.</li>
</ol>
3. Freezing NS for Long-term Storage
<ol>
	<li>When the NS culture becomes confluent, dissociate the cells as described in steps 2.1-2.4. Once the pellet has been re-suspended in HBSS, remove an aliquot and count the cells.</li>
	<li>Centrifuge cells at 300 x g for 4 min and remove as much as possible of the supernatant.</li>
	<li>Re-suspend the pellet in freezing media (heat-inactivated FBS, 10% DMSO). It is recommended to freeze between 1 x 106 and 3 x 106 cells per vial per mL.</li>
	<li>Divide the re-suspended cells in as many cryovials as needed and slowly cool down the vials by first transferring them to ice, then to -20 &#176;C, then to -80 &#176;C, and finally the next day to a liquid nitrogen storage container.</li>
</ol>
4. Fixation of Tumor NS
<ol>
	<li>Culture tumor NS in a T-25 flask for 2-3 days until cells are confluent (~ 3 x 106 cells). Collect tumor NS in a 15 mL conical tube from a least one T-25 confluent flask. Pellet at 300 x g for 5 min.</li>
	<li>Re-suspend the pellet in 1 mL of 10% formalin and keep at room temperature (RT) for 10 min. Add 5 mL of 1x HBSS and pellet the cells as done in step 3.2. Repeat this step one more time.</li>
	<li>Place the 15 mL conical tube containing the pellet on ice.</li>
</ol>
5. Paraffin Embedding of Tumor NS
<ol>
	<li>Re-suspend the washed NS pellet in 500 &#181;L of 2% warm liquid agarose and mix gently by pipetting.</li>
	<li>Immediately place the agarose-embedded NS on ice until the agarose polymerizes. Remove the agarose piece harboring the NS. Place the polymerized agarose piece with the NS into the cassette for tissue processing (Figure 2).</li>
	<li>Continue with tissue processing (using an automatic paraffin processor) and paraffin embedding (using an embedding station).</li>
</ol>
6. Sectioning of Paraffin-embedded NS
<ol>
	<li>Set the water bath to 42 &#176;C and insert the blade on the microtome carefully using two paint brushes to make sure it is leveled. Use this blade only for trimming.</li>
	<li>Place the paraffin block on the microtome. With the non-dominant hand, press down on the lever located on left-hand side that controls the thickness of each section. Press to the second stop that indicates a 30 &#181;m thickness.</li>
	<li>Begin trimming the block (i.e., removing excess paraffin) by turning the coarse hand wheel with the right hand until the surface of the sample is reached. At this point, release the lever that controls the trim thickness to either 10 &#956;m or 5 &#956;m. Stop trimming when the surface of the sample is reached.</li>
	<li>Fill an ice box with ice and add just enough water so that when the paraffin block is placed on ice, the water acts as a film around the paraffin block, protecting it from directly touching the ice. Incubate the paraffin block on ice for 20 min.</li>
	<li>Discard the old blade and insert a new one for sectioning. Dry block using gauze, then place it on the microtome.</li>
	<li>With the non-dominant hand, obtain a pair of curved forceps that will be used to pull the paraffin ribbon. Keep a damp paint brush near the right hand, which will be used to transfer the paraffin ribbon to the water bath. Begin to section by turning the coarse hand wheel with the right hand at a fast pace. Use the forceps in the left hand to hold the paraffin ribbon.</li>
	<li>Use the damp paint brush to transfer the paraffin ribbon to the water bath.</li>
	<li>Use the curve of the forceps to break apart the sections by superficially placing the forceps in between two paraffin sections, then pushing the two sections away gently. 
	NOTE: This motion should be entirely on the surface of the paraffin section. Do not press down on the section.</li>
	<li>Use the damp paint brush to push the separated paraffin sections onto positively charged adhesion microscope slides.</li>
	<li>Allow the slides to dry overnight inside a chemical hood before storing them in slide boxes. The storage of slides depends on the type of stain performed.</li>
</ol>
7. Immunohistochemistry (IHC) of Paraffin-embedded NS
<ol>
	<li>Melt paraffin by placing the slides in a metal rack and incubating them at 60 &#176;C for 20 min.</li>
	<li>Deparaffinize the slides by incubation in a rehydration train at RT: 3x xylene for 5 min, 100% 2x ethanol for 2 min, 95% 2x ethanol for 2 min, 70% 1x ethanol for 2 min, and 1x deionized water for 2 min. Hereon, keep the slides in tap water until antigen retrieval, as drying out will cause non-specific antibody binding.</li>
	<li>Incubate the slides in citrate buffer (10 mM citric acid, 0.05% non-ionic detergent, pH 6) at 125 &#176;C for 30 s and at 90 &#176;C for 10 s. Let them cool down, then rinse the slides 5 times with distilled water.</li>
	<li>Incubate slides at RT in 0.3% H2O2 for 30 min.</li>
	<li>Wash the slides 3 times in washing buffer (0.025% detergent in TBS) for 5 min with gentle agitation.</li>
	<li>Incubate the slides at RT with blocking solution (10% horse serum, 0.1% BSA in TBS) for 30 min.</li>
	<li>Incubate the slides overnight at 4 &#176;C in a humidified chamber with primary antibody prepared in dilution solution (0.1% BSA in TBS). Wash the slides 3 times in washing buffer for 5 min with gentle agitation.</li>
	<li>Incubate the slides at RT for 1 h with biotinylated secondary antibody prepared in dilution solution. Wash the slides 3 times in washing buffer for 5 min with gentle agitation.</li>
	<li>Incubate the slides at RT in the dark for 30 min with avidin-biotinylated horseradish peroxidase complex. Wash the slides 3 times in washing buffer for 5 min with gentle agitation.</li>
	<li>Incubate the slides with DAB-H2O2 substrate brands here for 2-5 min at RT.</li>
	<li>Rinse 3 times with tap water. Dip (1-2 s) the slides in hematoxylin solution to counterstain, and rinse them thoroughly in tap water until clearing.</li>
	<li>Dehydrate the slides as follows: incubate in distilled water for 2 min, dip 3 times in 80% ethanol, incubate in 95% ethanol 2 times for 2 min each, incubate in 100% ethanol 2 times for 2 min each, and finally in xylene 3 times for 3 min each.</li>
	<li>Coverslip the slides with a xylene-based mounting medium and let them dry on a flat surface at RT until they are ready for imaging.&#160; &#160; &#160; &#160;NOTE: If performing 3,3&#39;-diaminobenzidine (DAB) staining, the slides can be stored at RT. However, if immunofluorescence staining is performed, slides should be stored in the dark at -20 &#176;C to reduce photo-bleaching.</li>
</ol>

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The authors have nothing to disclose.

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.

<table><tbody><tr><td>Coated microtome blade HP35</td><td>Thermo Scientific</td><td>3150734</td><td></td></tr><tr><td>Microtome RM 2135</td><td>Leica</td><td>MR2135</td><td></td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma-aldrich</td><td>HT501128-4L</td><td></td></tr><tr><td>Rabbit polyclonal anti-ATRX,</td><td>Santa Cruz Biotechnology</td><td>sc-15408</td><td>IHC, 1:250 dilution</td></tr><tr><td>Rabbit polyclonal anti-Ki-67</td><td>Abcam</td><td>Ab15580</td><td>IHC, 1:1000 dilution</td></tr><tr><td>Rabbit polyclonal anti-OLIG2</td><td>Millipore</td><td>AB9610</td><td>IHC, 1:500 dilution</td></tr><tr><td>Goat polyclonal anti-rabbit biotin-conjugated</td><td>Dako</td><td>E0432</td><td>IHC, 1:1000 dilution</td></tr><tr><td>Vectastain Elite ABC HRP kit</td><td>Vector Laboratories Inc</td><td>PK-6100</td><td></td></tr><tr><td>BETAZOID DAB CHROMOGEN KIT</td><td>Biocare medical</td><td>BDB2004 L/price till 12/18</td><td></td></tr><tr><td>N-2 Supplement</td><td>ThermoFisher</td><td>17502048</td><td></td></tr><tr><td>N-27 Supplement</td><td>ThermoFisher</td><td>A3582801</td><td></td></tr><tr><td>Accutase&amp;reg; Cell Detachment Solution</td><td>Biolegend</td><td>423201</td><td></td></tr><tr><td>AGAROSE LE</td><td>GoldBio</td><td>A-201-1000</td><td></td></tr><tr><td>Genesee Sc. Corporation</td><td>Olympus 15 ml</td><td>21-103</td><td></td></tr><tr><td>Genesee Sc. Corporation</td><td>TC-75 treated Flask</td><td>25-209</td><td></td></tr><tr><td>Genesee Sc. Corporation</td><td>TC-25 treated Flask</td><td>25-207</td><td></td></tr><tr><td>DMEM/F12</td><td>Gibco</td><td>11330-057</td><td>NS media</td></tr><tr><td>HBSS</td><td>Gibco&lt;sup&gt;TM&lt;/sup&gt;</td><td>14175-103</td><td>balanced salt solution</td></tr><tr><td>C57BL/6</td><td>Taconic</td><td>B6-f</td><td>C57BL/6 mouse</td></tr></tbody></table>

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 ...

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JoVE Journal

Cancer Research

7.8K Views.  University of Michigan Medical School. Dit protocol is belangrijk omdat kan worden gebruikt om de aanwezigheid van glioom biomarker te bestuderen door middel van een methode die de 3D-structuur van tumorneuros behoudt. Het belangrijkste voordeel van dit protocol is dat de morfologie van neurosferen wordt gehandhaafd in vergelijking met cytospin methode waarbij cellen worden onderworpen aan stressvolle manipulatie en worden afgevlakt. Deze methode kan worden toegepast op andere weefselkweeksystemen waarin cellen in 3D worden gekwee...

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

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Isolation and Expansion of Human Glioblastoma Multiforme Tumor Cells Using the Neurosphere Assay

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in glioblastoma mutliforme (GBM), is to identify and isolate tumor initiating cell population(s) to investigate their role in tumor formation, progression, and recurrence. Understanding tumor initiating cell populations will provide clues to finding effective therapeutic approaches for these tumors. The neurosphere assay (NSA) due to its simplicity and reproducibility has been used as the method of choice for isolation and propagation of many of this tumor cells. This protocol demonstrates the neurosphere culture method to isolate and expand stem-like cells in surgically resected human GBM tumor tissue. The procedures include an initial chemical digestion and mechanical dissociation of tumor tissue, and subsequently plating the resulting single cell suspension in NSA culture. After 7-10 days, primary neurospheres of 150-200 &mu;m in diameter can be observed and are ready for further passaging and expansion.

This video protocol demonstrates the isolation and expansion of stem like cells from surgically resected human glioblastoma mutliforme (GBM) tumor tissue using the neurosphere assay culture method.

Stem-like cells have been isolated in tumors such as breast, lung, colon, prostate and brain. A critical issue in all these tumors, especially in ...

Neuroscience

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 ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Optimization of High Grade Glioma Cell Culture from Surgical Specimens for Use in  Clinically Relevant Animal Models and 3D Immunochemistry

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell cultures that preserve the genomic profile of the parental tumors, for use in patient specific in vitro and in vivo models, has the potential to revolutionize the preclinical development of new treatments for glioblastoma tailored to the molecular characteristics of each tumor.
Starting with fresh high grade astrocytoma tumors dissociated into single cells, we use the neurosphere assay as an enrichment method for cells presenting cancer stem cell phenotype, including expression of neural stem cell markers, long term self-renewal in vitro, and the ability to form orthotopic xenograft tumors. This method has been previously proposed, and is now in use by several investigators. Based on our experience of dissociating and culturing 125 glioblastoma specimens, we arrived at the detailed protocol we present here, suitable for routine neurosphere culturing of high grade astrocytomas and large scale expansion of tumorigenic cells for preclinical studies. We report on the efficiency of successful long term cultures using this protocol&#160;and suggest affordable alternatives for culturing dissociated glioblastoma cells that fail to grow as neurospheres. We also describe in detail a protocol for preserving the neurospheres 3D architecture for immunohistochemistry. Cell cultures enriched in CSCs, capable of generating orthotopic xenograft models that preserve the molecular signatures and heterogeneity of GBMs, are becoming increasingly popular for the study of the biology of GBMs and for the improved design of preclinical testing of potential therapies.

Protocol for propagation of dissociated high grade glioma surgical specimens in serum-free neurosphere medium to select for cells with cancer stem cell phenotype. For specimens that fail to grow as neurospheres, an alternative protocol is suggested. A paraffin embedding technique for maintaining the 3D neurosphere architecture for immunocytochemistry is described.

Glioblastomas, the most common and aggressive form of astrocytoma, are refractory to therapy, and molecularly heterogeneous. The ability to establish cell ...

Medicine

Spheroid Drug Sensitivity Screening in Glioma Stem Cell Lines

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens are performed on cells grown in a monolayer. However, these two-dimensional (2D) models are considered less accurate compared to three-dimensional (3D) spheroid cell models; this is especially true for glioma stem cell lines. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening of spheroid lines; mouse and human glioma stem cell lines are used as an example. This protocol describes a 3D spheroid drug sensitivity and synergy assay that can be used to determine if a drug or drug combination induces cell death and if two drugs synergize. Glioma stem cell lines are&#160;modified to express RFP. Cells are plated in low attachment round well bottom 96 plates, and spheres are allowed to form overnight. Drugs are added, and the growth is monitored by measuring the RFP signal over time using the Incucyte live imaging system,&#160;a fluorescence microscope embedded in the tissue culture incubator. Half maximal inhibitory concentration (IC50), median lethal dose (LD50), and synergy score are subsequently calculated to evaluate sensitivities to drugs alone or in combination. The three-dimensional nature of this assay&#160;provides a more accurate reflection of tumor growth, behavior, and drug sensitivities in vivo, thus forming the basis for further preclinical investigation.

In vitro drug sensitivity screens are important tools for discovering anti-cancer drug combinations. Cells grown in spheres activate different signaling pathways and are considered more representative of in vivo models than monolayer cell lines. This protocol describes a method for in vitro drug screening for spheroid lines.

In vitro drug sensitivity screens are important tools in the discovery of anti-cancer drug combination therapies. Typically, these in vitro drug screens ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within patient samples of glioblastoma, the deadliest and most invasive brain cancer. Not only are quantitative methods beneficial for accurately describing diseased tissues, they can also potentially contribute to more accurate prognosis, diagnosis, and the development of tissue-engineered systems and replacements. In glioblastoma, glial cells, such as microglia and astrocytes, have been independently correlated with poor prognosis based on pathologist grading. However, the state of these cells and other glial cell components has not been well-described quantitatively. This can be difficult due to the large processes that mark these glial cells. Furthermore, most histological analyses focus on the overall tissue sample or only within the bulk of the tumor, as opposed to delineating quantifications based on regions within the highly heterogeneous tissue. Here, we describe a method for identifying and quantitatively analyzing the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations in patient tumor resections and ImageJ to analyze percent coverage of staining for each glial population. With these techniques we are able to better describe the glial cells throughout regions of the glioma tumor microenvironment.

This protocol was developed to quantitatively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and ImageJ.

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

Immunohistochemical Staining of Biomarkers in Brain Tumor Neurosphere Sections

<a href='https://app.jove.com/v/58931/evaluation-biomarkers-glioma-immunohistochemistry-on-paraffin'>Source: N&#250;&#241;ez, F. J., et al. Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures.&#160;J. Vis. Exp. (2019).</a>This video demonstrates an immunohistochemical technique for labeling tumor biomarkers in paraffin-embedded brain tumor neurospheres. The neurosphere sections undergo paraffin removal and rehydration, followed by treatment to expose biomarker sites. Primary and secondary antibodies are then applied to label these biomarkers. The secondary antibodies are bound by avidin-conjugated peroxidase enzymes, which oxidize chromogenic substrates to indicate biomarker locations. Finally, nuclear counterstaining and slide preparation are completed for microscopic analysis.

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

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