This work was funded in part by a grant to D.S.G. from the National Cancer Institute (R03CA227312) and by a generous grant from the Lisa Dean Moseley Foundation. Live GBM specimens were obtained with patient consent through the Tissue Procurement Center of the Helen F. Graham Cancer Center and Research Institute. Funding to A.R. was provided by the National Center for Research Resources and the National Center for Advancing Translational Sciences, National Institutes of Health (UL1TR003107). Summer undergraduate research fellowships to N.P., A.L., Z.W., and K.S. were provided by the University of Delaware Undergraduate Research Program.

Using Chick Embryo Brain as a Model to Study Human Glioblastoma Cell Behavior

<ol>
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None of the authors have any conflicts of interest.

Critical steps in the protocol for the injection of cells into the midbrain (optic tectum) ventricle include not damaging the blood vessels in the chorioallantoic membrane in the egg or surrounding the embryo before and during injection, although the amnion membrane immediately surrounding the embryo can be gently pulled and held to position the head when injecting the cells into the midbrain. The amnion is relatively tough and can be pulled with fine forceps to position the head and hold it steady with one hand, for the injection of cells with the other hand into the optic tectum, which is the large, round structure in the middle of the brain. Generally, the viability of injected embryos ranges from 25% to 75%, depending upon unknown factors, and practically every embryo that survives contains at least a small tumor in the optic tectum. Critical steps in generating viable brain slices include blotting the tissue of excess liquid so that the agarose adheres to the brain during slicing and to keep tissue and slices cold until placed on the membrane insert. As different cell types form spheroids differently (in speed and size), the plated cell density on poly-HEMA plates and the length of time before harvesting spheroids should be optimized for each cell type.
The work here has not been subject to a formal longitudinal study of brain slice viability. Yang et al. used chick embryo brain slice cultures similar to the ones used here and showed good viability of the slices for at least 7 days16. Previous work showed that when OT tissue was kept in suboptimal media, many pyknotic nuclei appeared in the tissue, which did not occur in the slices in the work here. Additionally, when slices degenerate in suboptimal conditions, the blood vessels fragment and appear as rows of laminin-positive spheres (not shown). Thus, although the viability here has not been checked by methods such as electrophysiology or active caspase-3 expression, none of the indicators of cell death that were seen under suboptimal culture conditions appeared here.
The OT has been focused on for in vivo brain tumor experiments because it is the most easily injected region with the largest ventricle. At E5, which is the latest day that the embryo is small enough to remain accessible on top of the yolk, injections must be made into a ventricle, as all brain regions are nothing more than a thin ventricular zone. Nevertheless, these injections result successfully in embedded tumors with cells that invade the brain parenchyma. Sometimes, resulting tumors are found in the forebrain or cerebellum, but this is not common. Ex vivo slices of E15 optic tectum have been primarily used for experiments here, so that the ex vivo co-culture results can be correlated with the in vivo injection experiments. However, forebrain slices are also suitable and have a larger surface area and a very thin ventricle compared to the optic tectum, which might make the forebrain more suitable for ex vivo co-cultures that are not being correlated with in vivo injections.
It has been demonstrated here that in vivo injections, followed by tissue fixation, vibrating tissue slicer sectioning, and immunostaining for laminin and other markers, resulted in high-resolution images of GBM cells and GSCs in brain tissue in close proximity to blood vessels. The ability to determine the interrelationships between tumor cells and blood vessels was greatly facilitated by creating 3D volume renders from z-stacks of confocal optical sections using the confocal software and manufacturer&#39;s instructions. Time-lapse imaging using widefield fluorescence microscopy of GFP, mCherry, and DiD labeled cells was possible; however, migrating cells that were in close proximity to the highly fluorescent spheroids were sometimes obscured by the &#34;glow&#34; from the spheroid. This undesired effect can be somewhat minimized by carefully adjusting the exposure times for collecting widefield images. Time-lapse imaging using confocal z-stacks over time (4D) eliminated the out-of-focus glow from the spheroids and resulted in sharply defined migrating cells with a dark background. This was not described in the protocol, but was carried out similarly to widefield time-lapse imaging, which was performed while brain slices were on the transparent membrane inserts in a 6-well plastic plate. Although confocal time-lapse imaging results in markedly clearer images of individual cells and their behavior, a multi-point time-lapse experiment collecting z-stacks of 10 z-planes/point, at 10 min intervals over a 20 h period, is an extensive use of the scan head galvanometers. As this could significantly decrease the lifespan of the galvanometers, this method is used judiciously.
Although the chick embryo system is very suitable for both in vivo injection and ex vivo co-culture experiments that investigate GBM cell behavior, there are several limitations to this model system. As with any xenograft system, the environment in which human cells are implanted is not the human brain, but GBM cell behavior appears to mimic that in rodent models and in human patients. After performing in vivo injection experiments on E5, tumors are normally allowed to form for 10 days, until E15. This clearly is not enough time to study all aspects of tumorigenesis and cell invasion. However, it has been demonstrated here that solid tumors form in the brain parenchyma, cells interact and rearrange themselves within the tumor, and significant brain invasion occurs both along blood vessels and diffusely within this relatively short time period. Another limitation to the in vivo chick embryo system is that it is not suitable for drug or other treatments because of the large yolk and extraembryonic circulatory system that operates during chick embryo development. Topical liquid drug treatments would result in a highly variable and unknown concentration in the brain due to diffusion away from the embryo into the much larger yolk mass. Similarly, intravenous injection of drugs into the very delicate extraembryonic circulatory system would leak or diffuse out of the blood vessels and also result in unknown concentrations in the brain. This is one of the main reasons that the ex vivo slice culture method was adopted-so that not only could cell behavior be observed and tracked via time-lapse microscopy, but also so that treatments that have been successful in altering GBM cell behavior in a dish4 could be tested in a more relevant brain tissue environment.
The development of the chick embryo orthotopic brain tumor model system is seen as a significant addition to the systems and tools available for the study of GBM tumor formation and invasive cell behavior. Fertilized chicken eggs are likely to be readily available in most areas, they are inexpensive compared to rodents, there are no animal care costs, the embryos are very resilient and resistant to infection (i.e., most work is done on a bench top), the embryos are highly manipulable and can be grown in shell-less culture19, and chick embryos are not considered vertebrate animals and so do not require IACUC approval by NIH guidelines (institutional requirements may vary). Thus, these multiple advantages make the chick embryo system very attractive if one confines their questions and experiments to those that fall within its limitations. Multiple GBM cell studies have been performed by others using the chick embryo, but these almost exclusively have utilized the chorioallantoic membrane (CAM) of the embryo20,21,22,23,24,25,26,27,28,29 and limb bud30, and not the brain. There has also been a report implanting medulloblastoma into the chick brain on E231. Undoubtedly, the use of the chick embryo as an orthotopic xenograft model system, as described here, should yield results that are much more meaningful to human GBM tumor biology than studies using the CAM.
Although these studies have only begun to fully utilize the chick embryo brain tumor model system for studies of human GBM cell and GSC behavior, it is hoped that others will extend the uses and find further potential applications. One could imagine that not only will this system uncover mechanisms that regulate GBM tumor formation and cell behavior, but will also allow preclinical testing of specific drugs and substances on specific patients&#39; cells. For instance, if brain slice cultures were set up in advance, then tumor cells, pieces from surgical tumor resections, or patient-derived GBM organoids32 could be placed directly in ex vivo co-culture, and various treatments could be assessed in a matter of days. Similarly, dissociated patient cells could be injected directly into E5 midbrains in ovo to assess their ability to form tumors and invade brain parenchyma. Thus, it is hoped that the descriptions of the methods and representative results here will facilitate and encourage increased use of this highly underutilized system for brain cancer research.

In vitro studies of cancer cell behaviors are often used to dissect potential mechanisms that operate during the more complex behavior that is observed during tumor formation and cell invasion in in vivo xenograft models. For example, with glioblastoma (GBM), in vitro studies have uncovered mechanisms of how L1CAM potentially operates during tumor formation and brain invasion in a novel chick embryo xenograft brain tumor model1,2,3,4,5. Although in vitro and in vivo experiments complement each other in useful ways, they leave a substantial gap in how the results can be correlated. For instance, mechanistic analyses of GBM cell motility on a dish is a highly artificial situation, and in vivo xenograft models can only reveal static time point or endpoint analyses of tumor formation and cell behavior. In vivo studies using either rodents or chick embryos do not easily lend themselves to monitoring cell behavior while the cells invade brain tissue in these xenograft models. Nevertheless, the chick embryo xenograft model has demonstrated that the adhesion protein L1CAM plays a stimulatory role in the invasive ability of human T98G GBM cells2,5.
A suitable solution to this problem can be reached by bridging both in vivo and in vitro methods using an organotypic brain slice culture model, referred to as an ex vivo model. In this ex vivo model, live brain tissue can be maintained at a thickness of several hundred microns for up to a few weeks, making it possible to implant cancer cells, observe their behavior in actual tissue over time, and then perform a more detailed marker analysis at the endpoint of the experiment.
A popular organotypic slice culture method has been to culture a several hundred micron-thick brain slice on top of a translucent or transparent porous membrane, leaving the tissue exposed to air, yet allowing nutrient media to sustain the tissue from below the membrane (refer to Stoppini et al.6). Different variations of this method have been used for different studies, including using different media or different membrane inserts. Different membrane inserts include a 30 mm diameter, porous (0.4 &#181;m) membrane insert in a 35 mm culture dish6, and cell culture inserts (0.4 &#181;m) for 6-well plates7. Different media include 50% MEM/HEPES + 25% heat-inactivated horse serum + 25% Hanks balanced salt solution (HBSS)8, 50% reduced serum media + 25% horse serum + 25% HBSS9, as well as others. If a translucent or transparent membrane is used along with fluorescently labeled GBM cells, then such cultures can be imaged from below using an inverted widefield or confocal fluorescence microscope10,11,12,13,14,15.
While many in vivo orthotopic brain tumor xenograft and ex vivo organotypic brain slice culture models have been established using rodents, as cited above, the chick embryo (Gallus gallus) has been underutilized for these purposes. However, the chick embryo has been demonstrated to be capable of being used as an in vivo orthotopic xenograft model for the study of both human and rat glioma invasion1,2,5. Xenografted cells into chick embryo brains have exhibited invasion patterns similar to those observed in rodent models, further supporting the use of chick embryos as an in vivo model for GBM tumor cell analysis. Chick embryos also are inexpensive, can be more easily maintained than rodents (i.e., in their egg shells in a lab incubator), and are much easier to work with, making them an attractive option for short-term in vivo GBM studies. A recent article has described the use of chick embryo brain slice cultures for the formation and growth of axons during normal brain development where the slices were viable for at least 7 days16. However, the use of such chick embryo brain slice cultures for ex vivo analysis of GBM cell behavior in a tissue environment is lacking. In this article, both the transplantation of human GBM cells and GBM stem cells (GSCs) into the early chick embryo brain in vivo, as well as the introduction of GBM cells onto live chick embryo brain slice cultures ex vivo, are described. Some representative examples of the resulting tumors and cell invasion patterns obtained from these preparations are also provided.

<table><tbody><tr><td>1 cm x 1 cm square hole paper punch</td><td>Birabira</td><td>N/A</td><td></td></tr><tr><td>1 mm biopsy punch pen</td><td>Robbins Instruments</td><td>20335</td><td></td></tr><tr><td>6 well insert plate (Corning Transwell)</td><td>Millipore Sigma</td><td>CLS3450</td><td></td></tr><tr><td>9&quot; Disposable Pasteur Pipets</td><td>Fisher Scientific</td><td>13-678-20C</td><td></td></tr><tr><td>15 mL centrifuge tubes</td><td>Fisher Scientific</td><td>05-539-12</td><td></td></tr><tr><td>24 well plate</td><td>Corning Costar</td><td>3526</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>Fisher Scientific</td><td>05-539-9</td><td></td></tr><tr><td>Agar</td><td>Fisher BioReagents</td><td>BP1423-500</td><td>for embedding fixed brains</td></tr><tr><td>Alexafluor 488-conjugated GAM IgG</td><td>Jackson Immunoresearch</td><td>115-605-146</td><td></td></tr><tr><td>Alexafluor 647-conjugated GAM IgG</td><td>Jackson Immunoresearch</td><td>115-545-146</td><td></td></tr><tr><td>Aluminum foil</td><td>ReynoldsWrap</td><td>N/A</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma Aldrich</td><td>A-9518</td><td></td></tr><tr><td>anti-integrin alpha-6 monoclonal antibody GOH3</td><td>Santa Cruz Biotechnology</td><td>sc-19622</td><td></td></tr><tr><td>anti-L1CAM monoclonal antibody UJ127</td><td>Santa Cruz Biotechnology</td><td>sc-53386</td><td></td></tr><tr><td>anti-laminin monoclonal antibody</td><td>Developmental Studies Hybridoma Bank</td><td>3H11</td><td></td></tr><tr><td>anti-nestin monoclonal antibody 10c2</td><td>Santa Cruz Biotechnology</td><td>sc-23927</td><td></td></tr><tr><td>anti-Sox2 monoclonal antibody E-4</td><td>Santa Cruz Biotechnology</td><td>sc-365823</td><td></td></tr><tr><td>B27 supplement without vitamin A</td><td>GIBCO</td><td>17504-044</td><td></td></tr><tr><td>bisbenzimide (Hoechst 33258)</td><td>Sigma-Aldrich</td><td>B2883</td><td>nuclear stain</td></tr><tr><td>Cell culture incubator</td><td>Forma</td><td></td><td>standard humidified CO&lt;sub&gt;2&lt;/sub&gt; incubator</td></tr><tr><td>Centrifuge</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon Instruments</td><td>C2si+</td><td>With custom-made cell incubator chamber</td></tr><tr><td>Confocal microscope objective lenses</td><td>Nikon Instruments</td><td></td><td>Plan Apo lenses, except S Plan Fluor ELWD 20x 0.45 NA objective lens for confocal time-lapse imaging</td></tr><tr><td>Confocal microscope software</td><td>Nikon Instruments</td><td>NIS Elements</td><td>Version 5.2</td></tr><tr><td>Curved foreceps</td><td>World Precision Intruments</td><td>504478</td><td></td></tr><tr><td>Curved scissors</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Curved spatula</td><td>Fisher Scientific</td><td>14-375-20</td><td></td></tr><tr><td>Cyanoacrylate glue</td><td>Krazy Glue</td><td>KG-585 12R</td><td></td></tr><tr><td>D-Glucose</td><td>Millipore Sigma</td><td>G8270</td><td></td></tr><tr><td>DiD far red fluorescent dye</td><td>Invitrogen</td><td>V22887</td><td>Vybrant DiD</td></tr><tr><td>DMEM</td><td>Sigma Aldrich</td><td>D5671</td><td></td></tr><tr><td>DMEM/F12</td><td>Sigma Aldrich</td><td>D8437</td><td></td></tr><tr><td>DMSO</td><td>Sigma Aldrich</td><td>D4540</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate buffered saline (PBS)</td><td>Sigma Aldrich</td><td>P5493-1L</td><td></td></tr><tr><td>egg incubator</td><td>Humidaire</td><td></td><td></td></tr><tr><td>electrical tape (10 mil thick/254 &amp;micro;m)</td><td>Scotch</td><td>N/A</td><td></td></tr><tr><td>Ethanol 200 proof</td><td>Decon Laboratories</td><td>2701</td><td></td></tr><tr><td>Fast green FCF dye</td><td>Avocado Research Chemicals</td><td>16520</td><td></td></tr><tr><td>FBS</td><td>Gemini Bio-products</td><td>900-108</td><td></td></tr><tr><td>filter paper</td><td>Fisher Scientific</td><td></td><td></td></tr><tr><td>Gauze</td><td>Dynarex</td><td>3353</td><td></td></tr><tr><td>Glass Capillaries for microinjection</td><td>World Precision Instruments</td><td>TW100-4</td><td></td></tr><tr><td>Glycerol</td><td>Fisher BioReagents</td><td>BP228-1</td><td>for mounting media</td></tr><tr><td>GSCs (human glioblastoma stem cells)</td><td>Not applicable</td><td></td><td>Isolated from patient GBM specimens in Galileo laboratory in GSC media and then transduced with a GFP encoding lentiviral vector.&amp;nbsp; Cells used were between passage 10 and 30.</td></tr><tr><td>Hanks Balanced Salt Solution (HBSS)</td><td>Corning</td><td>21-020-CV</td><td></td></tr><tr><td>Hemacytometer</td><td>Hausser scientific</td><td></td><td></td></tr><tr><td>Heparin</td><td>Fisher Scientific</td><td>BP2524-100</td><td></td></tr><tr><td>HEPES buffer</td><td>Sigma Aldrich</td><td>H0887</td><td></td></tr><tr><td>Horse Serum (HI)</td><td>Gibco</td><td>26050-088</td><td></td></tr><tr><td>Human FGF-2</td><td>BioVision</td><td>4037-1000</td><td></td></tr><tr><td>Human TGF-&amp;alpha;</td><td>BioVision</td><td>4339-1000</td><td></td></tr><tr><td>Inverted phase contrast microscope</td><td>Nikon Instruments</td><td>TMS</td><td>for routine viewing of cultured cells</td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>BP366</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P284</td><td></td></tr><tr><td>Laboratory film</td><td>Parafilm</td><td></td><td></td></tr><tr><td>Labquake Shaker</td><td>LabIndustries</td><td>T400-110</td><td></td></tr><tr><td>L-Glut:Pen:Strep</td><td>Gemini Bio-products</td><td>400-110</td><td></td></tr><tr><td>Low-melt agarose</td><td>Fisher Scientific</td><td>BP1360</td><td>for embedding live brains</td></tr><tr><td>Matrix</td><td>Corning Matrigel</td><td>354234</td><td></td></tr><tr><td>Medium 199</td><td>GIBCO</td><td>11150-059</td><td></td></tr><tr><td>MEM</td><td>Corning</td><td>10-010-CV</td><td></td></tr><tr><td>Metal vibratome block</td><td></td><td></td><td></td></tr><tr><td>Micropipette tips (20, 200, 1,000 &amp;micro;L)</td><td>Fisherbrand</td><td></td><td></td></tr><tr><td>Micropipettors (20, 200, 1,000 &amp;micro;L)</td><td>Gilson</td><td></td><td></td></tr><tr><td>Microscope Coverglass (no. 1.5 thickness)</td><td>Fisherbrand</td><td>12544A</td><td></td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; + H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Fisher Scientific</td><td>S369</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>BP328</td><td></td></tr><tr><td>Normal goat serum</td><td>Millipore Sigma</td><td>526-M</td><td></td></tr><tr><td>N-propyl gallate</td><td>Sigma Aldrich</td><td>P3130</td><td>for mounting media</td></tr><tr><td>Parafilm</td><td>Parafilm</td><td></td><td></td></tr><tr><td>Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td></td></tr><tr><td>PBS</td><td>Sigma Aldrich</td><td>P5493-1L</td><td></td></tr><tr><td>Pencil</td><td></td><td></td><td></td></tr><tr><td>Plain Microscope slides</td><td>Fisherbrand</td><td>12-550-A3</td><td></td></tr><tr><td>Plastic 35 mm Petri dish</td><td>Becton Dickinson</td><td>351008</td><td></td></tr><tr><td>pneumatic picopump</td><td>World Precision Intruments</td><td>PV830</td><td></td></tr><tr><td>Poly(2-hydroxyethyl methacrylate)&amp;nbsp; (poly-HEMA)</td><td>Sigma Aldrich</td><td>P-3932</td><td></td></tr><tr><td>razor blade- double edge</td><td>PACE</td><td></td><td>for cutting fixed brain slices</td></tr><tr><td>sapphire knife</td><td>Delaware Diamond Knives</td><td></td><td>for cutting live brain slices</td></tr><tr><td>Scalpel</td><td>TruMed</td><td>1001</td><td></td></tr><tr><td>Sodium cacodylate buffer 0.2 M pH 7.4</td><td>Electron Microscopy Sciences</td><td>11652</td><td></td></tr><tr><td>Specimen chamber for vibratome</td><td>custom-made</td><td></td><td></td></tr><tr><td>Stereo Dissecting Microscope</td><td>Nikon Instruments</td><td>SMZ1500</td><td>Equipped with epifluorescence</td></tr><tr><td>straight foreceps</td><td>World Precision Intruments</td><td>500233</td><td></td></tr><tr><td>straight scissors</td><td>Fine Science Tools</td><td></td><td></td></tr><tr><td>Sucrose</td><td>Mallinckrodt</td><td>7723</td><td></td></tr><tr><td>Time-lapse fluorescence microscope (widefield fluorescence)</td><td>Nikon Instruments</td><td>TE2000-E</td><td>With custom-made cell incubator chamber (see Fotos et al., 2006)</td></tr><tr><td>Tissue culture dish polystyrene 100 mm</td><td>Thermo Fisher Scientific</td><td>130182</td><td>for cell culturing</td></tr><tr><td>Tissue culture dish polystyrene 60 mm</td><td>Becton Dickinson</td><td>353004</td><td>for cell culturing</td></tr><tr><td>Transfer pipette</td><td>American Central Scientific Co.</td><td>FFP011</td><td></td></tr><tr><td>Transparent tape</td><td>Scotch</td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Sigma Aldrich</td><td>T-8787</td><td></td></tr><tr><td>Trypsin (0.25%) + 2.21 mM EDTA</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>U-118 MG human GBM cell line</td><td>ATCC</td><td>HTB-15</td><td>Cells were transduced with a lentiviral vector encoding the entire ectodomain sequence of the L1CAM adhesion protein and then with lentiviral vector pUltra-hot encoding mCherry.&amp;nbsp; Passage numbers are unknown.</td></tr><tr><td>Vacuum pump</td><td>Cole-Parmer</td><td>EW-07532-40</td><td>&quot;Air Cadet&quot;</td></tr><tr><td>Vibrating tissue slicer</td><td>Vibratome</td><td>3000</td><td>for cutting live and fixed brain slices</td></tr></tbody></table>

using the chick embryo brain as a model for in vivo and ex vivo analyses of human glioblastoma cell behavior

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick embryo has been extended for studying the formation of human glioblastoma (GBM) brain tumors in vivo and the invasiveness of tumor cells into surrounding brain tissue. GBM tumors can be formed by injection of a suspension of fluorescently labeled cells into the E5 midbrain (optic tectum) ventricle in ovo. 
Depending on the GBM cells, compact tumors randomly form in the ventricle and within the brain wall, and groups of cells invade the brain wall tissue. Thick tissue sections (350 &#181;m) of fixed E15 tecta with tumors can be immunostained to reveal that invading cells often migrate along blood vessels when analyzed by 3D reconstruction of confocal z-stack images. Live E15 midbrain and forebrain slices (250-350 &#181;m) can be cultured on membrane inserts, where fluorescently labeled GBM cells can be introduced into non-random locations to provide ex vivo co-cultures to analyze cell invasion, which also can occur along blood vessels, over a period of about 1 week. These ex vivo co-cultures can be monitored by widefield or confocal fluorescence time-lapse microscopy to observe live cell behavior. 
Co-cultured slices then can be fixed, immunostained, and analyzed by confocal microscopy to determine whether or not the invasion occurred along blood vessels or axons. Additionally, the co-culture system can be used for investigating potential cell-cell interactions by placing aggregates of different cell types and colors in different precise locations and observing cell movements. Drug treatments can be performed on ex vivo cultures, whereas these treatments are not compatible with the in ovo system. These two complementary approaches allow for detailed and precise analyses of human GBM cell behavior and tumor formation in a highly manipulatable vertebrate brain environment.

Watch this Scientific Journal Video about Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior at JoVE.com

Author Spotlight: Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior  

Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis.

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior

The chick embryo has been an ideal model system for the study of vertebrate development, particularly for experimental manipulations. Use of the chick ...

using-chick-embryo-brain-as-model-for-vivo-ex-vivo-analyses-human

Research

JoVE Journal

Neuroscience

3.5K Views.  University of Delaware. Chick embryos are used for studying human glioblastoma (GBM) brain tumors in ovo and in ex vivo brain slice co-cultures. GBM cell behavior can be recorded by time-lapse microscopy in ex vivo co-cultures, and both preparations can be analyzed at the experimental endpoint by detailed 3D confocal analysis. 

Video: Author Spotlight: Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior  

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Ex vivo Culturing of Whole, Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab1 has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)2, was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine3 causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.
A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly4, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern5. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches6, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B17, and that is dependent on the activity of the ubiquitin-proteasome system6. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.

Ex vivo Culturing of Whole, Developing Drosophila Brains

This article describes a method by which one can mimic in vivo development of the Drosophila mushroom body in an ex vivo culture system.

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite significant advances in the genetics of these tumors, how GBM cells invade the healthy brain parenchyma is not well understood. Notably, it has been shown that GBM cells invade the peritumoral space via different routes; the main interest of this paper is the route along white matter tracts (WMTs). The interactions of tumor cells with the peritumoral nervous cell components are not well characterized. Herein, a method has been described that evaluates the impact of neurons on GBM cell invasion. This paper presents an advanced co-culture in vitro assay that mimics WMT invasion by analyzing the migration of GBM stem-like cells on neurons. The behavior of GBM cells in the presence of neurons is monitored by using an automated tracking procedure with open-source and free-access software. This method is useful for many applications, in particular, for functional and mechanistic studies as well as for analyzing the effects of pharmacological agents that can block GBM cell migration on neurons.

Here, we present an easy-to-use co-culture assay to analyze glioblastoma (GBM) migration on patterned neurons. We developed a macro in FiJi software for easy quantification of GBM cell migration on neurons, and observed that neurons modify GBM cell invasive capacity.

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...

Cancer Research

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of existing mouse lines for genetic cell lineage tracing: a tamoxifen-inducible Cre line and a Cre reporter line expressing dsRed upon Cre-mediated recombination. By using a relatively low level of tamoxifen, we were able to induce recombination in a small number of cells, permitting us to follow individual cell divisions. Additionally, we observed the transcriptional response to Sonic Hedgehog (Shh) signaling using an Olig2-eGFP transgenic line 1-3 and we monitored formation of cilia by infecting the cultured slice with virus expressing the cilia marker, Sstr3-GFP 4. In order to image the neuroepithelium, we harvested embryos at E8.5, isolated the neural tube, mounted the neural slice in proper culturing conditions into the imaging chamber and performed time-lapse confocal imaging. Our ex vivo live imaging method enables us to trace single cell divisions to assess the relative timing of primary cilia formation and Shh response in a physiologically relevant manner. This method can be easily adapted using distinct fluorescent markers and provides the field the tools with which to monitor cell behavior in situ and in real time.

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

Here we develop the tools necessary for ex vivo live imaging to trace single cell divisions in the mouse E8.5 neuroepithelium

We developed a system that integrates live imaging of fluorescent markers and culturing slices of embryonic mouse neuroepithelium. We took advantage of ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Medicine

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution has been a long sought tool for neuroscientists in the systems-level search for the neural circuitry governing complex behavioral states. Optogenetics is a cutting-edge tool that is revolutionizing the field of neuroscience and represents one of the first systematic approaches to enable causal testing regarding the relation between neural signaling events and behavior. By combining optical and genetic approaches, neural signaling can be bi-directionally controlled through expression of light-sensitive ion channels (opsins) in mammalian cells. The current protocol describes delivery of specific wavelengths of light to opsin-expressing cells in deep brain structures of awake, freely-moving rodents for neural circuit modulation. Theoretical principles of light transmission as an experimental consideration are discussed in the context of performing in vivo optogenetic stimulation. The protocol details the design and construction of both simple and complex laser configurations and describes tethering strategies to permit simultaneous stimulation of multiple animals for high-throughput behavioral testing.

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Optogenetics has become a powerful tool for use in behavioral neuroscience experiments. This protocol offers a step-by-step guide to the design and set-up of laser systems, and provides a full protocol for carrying out multiple and simultaneous in vivo optogenetic stimulations compatible with most rodent behavioral testing paradigms.

The ability to probe defined neural circuits in awake, freely-moving animals with cell-type specificity, spatial precision, and high temporal resolution ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

Developmental Biology

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Using the Chick Embryo Brain as a Model for In Vivo and Ex Vivo Analyses of Human Glioblastoma Cell Behavior

Summary

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Chapters in this video

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Ex vivo Culturing of Whole, Developing Drosophila Brains

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

Ex vivo Live Imaging of Single Cell Divisions in Mouse Neuroepithelium

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

In vivo Optogenetic Stimulation of the Rodent Central Nervous System

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs